Topical steroidal formulations

ABSTRACT

The present invention relates to formulations of poorly water soluble pharmaceutical agents of Formula I and II. The present invention also relates to compositions containing compounds of Formula I or II, and glucocorticoids, and methods for reducing side effects from glucocorticoid treatment by co-administration of compounds of Formula I and II. The compositions herein are useful for the treatment of diabetes and obesity related diseases including metabolic syndrome.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/076,784, filed on Jun. 30, 2008. The entire teachings of the aboveapplication are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant CA-12227from National Cancer Institute. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Some of the compounds of Formulae I and II are generally useful asanti-cancer, anti-obesity, anti-diabetic, anti-coronary agents,anti-aging agents, anti-hypolipidemic agents and anti-autoimmune agents.(Schwartz et. al., U.S. Pat. Nos. 5,656,621; 4,898,694; 5,001,119,5,028,631; 5,157,031, 5,696,106; 5,700,793; 5,714,481, 5,804,576). Inparticular, 16α-fluoro-5-androsten-17-one (fluasterone) has been shownto have reduced androgenic effects while maintaining the therapeuticeffects in comparison to dehydroepiandrosterone (DHEA). (McCormick et.al., Carcinogenesis 2007 28(2):398-403). Fluasterone has been shown toimprove recovery from traumatic brain injury in rat model of traumaticbrain injury. (Malik et. al. Journal of Neurotrauma 20:463-476, 2003).Furthermore, Fluasterone is also known to inhibit collagen-inducedarthritis in mice, indicating efficacy against rheumatoid arthritis.(Offfier et. al, Clinical Immunology, 110(2), 2004, 181-190; Williams J.R. et. al., Methods in Molecular Medicine, 2004, 98, 207-216).Fluasterone treatment inhibits the acute inflammatory and hyperplasticeffects of 12-O-tetradecanoylphorbol-13-acetate (TPA) induced skinpapilloma formation. (Schwartz et. al. Cancer Res. 48:4817 4822, 1988;Schwartz et. al., Carcinogenesis 10:1809-1813, 1989; Pashko et. al.Carcinogenesis 12:2189-2192, 1991; Hastings et. al., Carcinogenesis9:1099-102, 1988).

Problems associated with steroidal compositions include theandrogenicity and poor oral availability associated with them. Forexample, clinical trials with DHEA are encumbered by the high oral dosesrequired as well as the conversion of DHEA into active androgens. Theuse of less androgenic congeners as well as non-oral formulations mayfacilitate testing of this class of compounds. (Schwartz et. al., AgeingResearch Reviews, 3(2), 2004, 171-187. The formulations herein addresssolubility problems associated with insoluble pharmaceutical agents, andallows the preparation of topical formulations of compositions with lowandrogenicity.

Compounds of Formula I and II are useful against a wide variety ofdiseases, including cancer, diabetes and metabolic syndrome. Forexample, fluasterone due to its reduced androgenic effect would beuseful for chemoprevention.

Glucocorticoids are a widely used class of steroid hormones, but areassociated with significant side effects. Glucocorticoids arecharacterized by an ability to bind with the glucocorticoid receptor.(Thomas et. al., U.S. Patent Publication No. 20060069071). Topicalglucocorticoids are well known to cause local skin atrophy (thinning ofthe skin), purpura (bruised-appearing skin), striae (“stretch marks”),tolerance and “addiction syndrome”. Combineddehydroepiandrosterone-glucocorticoid compositions have been describedas effective pharmaceutical treatments for dermatitis. (Thomas et. al.,WO2006/036484). Furthermore, DHEA is known to have antiglucocorticoideffects in the brain. (R. Morfin, DHEA and the brain, Wiley, 2002,52-53). However, DHEA is known to have androgenic side effects. As such,it would be advantageous to have compositions with reduced androgenicityto reduce side effects associated with glucocorticoids.

Inhibition of G6PDH was believed to be a critical mechanism by whichDHEA and DHEA analogs reduce inflammation and oxygen free radicalformation. (Schwartz et. al., Ageing Res. Reviews. 3:171-187, 2004). Theeffectiveness of fluasterone against TPA-induced skin papillomaformation was believed to be mediated by the inhibition of glucose6-phosphate dehydrogenase (G6PDH) with a consequent lowering of NADPHlevels and reactive oxygen formation. (Schwartz et. al., Cancer Lett.168:7-14, 2001). Without being bound by any mechanism, the data hereinindicate that G6PDH inhibition may not be the mechanism by which thesesteroids produce an anti-glucocorticoid effect. Compound 8356(16α-fluoro-5α-androstan-17-one) is a more potent G6PDH inhibitor thanfluasterone yet it is about ⅛ as active in protecting againstdexamethasone. (Schwartz et. al., Cancer Res., 48:4817 4822, 1988).However, due to poor water solubility oral bioavailability offluasterone has required very high dosage levels. Nanosized fluasteroneformulations were shown to have better bioavailability than unmilled andmicronized formulations. (Janjikhel, et. al., AAPS Annual MeetingAbstracts, 2002). Even nanosized formulations required very high dosages(100 mg/kg) for oral delivery.

Thus, fluasterone is difficult to formulate, particularly for topicaladministration, due at least in part, to its very low water solubility.Therefore, a need exists to provide new formulations for theadministration of fluasterone.

SUMMARY OF THE INVENTION

The present invention generally relates to formulations of poorly watersoluble agents for pharmaceutical or veterinary use. The formulations ofthe present invention can be used for oral, topical, sublingual, buccal,intradermal, nebulization or inhalation methods of administration ofpoorly water soluble pharmaceutical agents. The present inventionfurther relates to the delivery of poorly water soluble compounds ofFormulae I and II.

wherein

R₁, R₂, R₃, R₄, R₅, R₆, and R₇ are each individually hydrogen or loweralkyl or hydroxyl or alkoxy having 1 to 5 carbons;

X is halogen, hydroxyl, hydrogen, lower alkyl or lower alkoxy having 1to 5 carbons;

Z is hydrogen or lower alkyl or alkoxy having 1 to 5 carbons, and

n is 1 or 2;

wherein at least one of X and Z is other than hydrogen.

The formulations of the present invention are particularly useful fortopical use because they dry quickly while providing an emollienteffect. One of the advantages of the co-solvent used herein is its watersolubility, which enables one to readily wash it off one's skin.Furthermore, the formulations herein are useful for topical (includingbuccal and sublingual), vaginal and parenteral (including subcutaneous,intramuscular, intradermal, intrathecal and epidural) delivery ofcompounds with poor oral availability.

Another aspect of the present invention is the use of compounds offormula I and II to alleviate the side effects of corticosteroidtreatments. This invention in particular relates to co-administration offluasterone with glucocorticoids. Applicants have surprisingly foundthat fluasterone has high potency when compared to several other relatedsteroids, including DHEA, in protecting mice againstdexamethasone-induced thymic involution. This marked anti-glucocorticoidactivity can be particularly useful to treat cancer patients.

Another aspect of the invention is the preparation of micronized andnano-sized particles of formulae I and II, particularly fluasterone.These smaller sized particles of compounds of formulae I and II can beparticularly useful for topical applications. Such micronized andnano-sized particles of compounds of formulae I and II, particularlyfluasterone, is useful for the treatment of diabetes, obesity andmetabolic syndrome.

The compositions herein are useful for the treatment of diabetes,obesity related diseases and metabolic syndromes. The compositionsherein are further useful for the treatment of arthritis, includingrheumatoid arthritis and osteoarthritis, skin diseases includingdermatitis, atherosclerosis and cancer. In addition to itschemopreventive activity, fluasterone protects against neoplasticdevelopment in a number of tissues including the prostate and otherepithelial tissues. The compositions herein are particularly useful asanti-cancer, anti-proliferative agents against tumors and cancersrelating to prostate, mammary gland, skin, colon, lung, lymphaticsystem, liver and rat thyroid.

Topical applications of the instant application are particularly usefulfor the treatment of osteoarthritis of the joints. In particular thehand, foot, thumb, forearm, knees, hip, jaw and elbow joints can betreated locally with topical applications containing compounds offormula I and II. Metacarpophalangeal, temporomandibular,trapeziometacarpal, metatarsal, carpus, ginglymus and sternoclaviculararticulation joints can be treated with compositions of the instantapplication.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

This invention is based, at least in part, of the discovery thatformulations of fluasterone, and related compounds, can be made bycombining the compound with organic alcohols, such as ethanol, andpolysorbates, such as a Tween compound (e.g. Tween 80) and solubilizingthe compound. Additional components or excipients can be added to theresulting solution, such as a viscosity enhancing agent or a gellingagent. Formulations made according to the invention can be topicallyapplied, achieving good to excellent absorption without creating anuncomfortable feeling on the skin.

In one aspect, the present invention relates to formulations containingcompounds of Formula I and II.

wherein

R₁, R₂, R₃, R₄, R₅, R₆, and R₇ are each individually hydrogen or loweralkyl or hydroxyl or alkoxy having 1 to 5 carbons;

X is halogen, hydroxyl, hydrogen, lower alkyl or lower alkoxy having 1to 5 carbons;

Z is hydrogen or lower alkyl or alkoxy having 1 to 5 carbons, and

n is 1 or 2;

wherein at least one of X and Z is not hydrogen.

The stereochemistry of various substituents are designated as being inthe α-position by means of a broken line

joining the substituent to the steroid nucleus. The substituents aredesignated as being in the β-position by means of a solid line (-)joining the substituent to the steroid nucleus. In those cases in whichthe substituents may be either in the α or β positions, the substituentsare indicated as being joined to the steroid nucleus by a broken lineand a solid line placed side to side. The compounds of formula I and IIcan be prepared by methods described in U.S. Pat. Nos. 5,656,621;4,898,694; 5,001,119, 5,028,631; 5,157,031; 5,696,106; 5,700,793;5,714,481, the entire contents of which are incorporated by referenceherein.

The following compounds of formula I and II are preferred;3β-methyl-16α-fluoro-5-androsten-17-one, 16α-methyl-5-androsten-17-one,3β-methyl-5-androsten-17-one, 3β-methyl-16α-methyl-5-androsten-17-one,3β-methyl-16α-chloro-5-androsten-17-one, 16α-fluoro-5α-androstan-17-one(8356), 7α-hydroxyfluasterone, 16α-fluoro-5-androsten-17β-ol,16α-hydroxy-5-androsten-17-one and 16α-fluoro-5-androsten-17-one(fluasterone) is more preferred.

Compounds of formula I and II, including fluasterone suffer from poorsolubility in water and require high oral doses in animals to produceefficacy. The lowest effective oral dose of fluasterone which abolishes12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated epidermalhyperplasia in mouse skin was determined to be 200 mg/kg, whereas whenadministered by subcutaneous injection, the lowest effective dose was2.5-5 mg/kg. The same dose relationship was observed in BKS. Cg-m⁺/₊Lepr^(db) diabetic mice for efficacy in lowering fasting plasma glucoseand triglyceride levels. Orally-administered ¹⁴C-Fluasterone undergoesextensive first-pass hepatic and/or gastrointestinal metabolism, thusnecessitating high oral doses to achieve efficacy.

In dose-response experiments the anti-hyperplastic efficacy offluasterone in TPA-treated mouse epidermis to determine bioavailabilityfollowing different routes of administration was measured. Mice weretreated with a single dose of fluasterone (either orally,subcutaneously, or transdermally) followed by a single application ofTPA to the shaved back one hour latter. In case of transdermaladministration, fluasterone is applied in a topical formulation to theshaved abdomen to allow for systemic absorption to the back of theanimal. Twenty hours after TPA application, the mice are injected with60 μCi of [3H] thymidine, sacrificed 20 minutes later, and a 2×2 cm2 ofskin from the shaved back is excised and processed for the determinationof the amount of [3H] thymidine incorporated per unit of epidermal DNA.(Hennings et. al., Cancer Res. 28:543-552, 1968).

Topical application of TPA produces a 2-to-4-fold increase in both thecpm of [³H] thymidine per μg epidermal DNA as well as a 2-to-4-foldincrease in the epidermal DNA content per 2×2 cm2 of skin. Thishyperplastic effect of TPA, as well as its reversal by fluasterone, hasbeen confirmed by histological examination of the mouse skin (Schwartzand Pashko, Cancer Lett. 168:7-14, 2001).

The lowest dose of orally-administered fluasterone which abolishesTPA-stimulated epidermal [³H] thymidine incorporation and epidermal DNAcontent is 200 mg/kg. This is also the lowest reported oral dose whichinhibits TPA-promoted skin tumor promotion (Schwartz, et al.Carcinogenesis 10:1809-1813, 1989)). This is also the lowest oral dosewhich lowers fasting plasma glucose in diabetic mice. In the TPA test asthe oral dose is increased beyond 200 mg/kg, there is an increase in therate of [³H] thymidine incorporation per unit of DNA, whereas theepidermal DNA content per 2×2 cm² of skin remains depressed. This“U-shaped” dose-response is seen with all routes of administration andpresumably results from a decrease in the endogenous thymidine pool sizeas a consequence of G6PDH inhibition (Nyce, J. W. Cellular and molecularaspects of 1,2-dimethylhydrazine-induced murine colonic adenocarcinomas,and their inhibition by dehydroepiandrosterone. Ph.D Thesis, TempleUniversity, pp 35-37, University Microfilms Int., Ann Arbor, Mich.,1983).

Transdermal administration of fluasterone was as potent as subcutaneousinjection in abolishing TPA-stimulated hyperplasia.

Compositions according to the invention are especially advantageous forthe topical administration to the skin of human subjects of fluasteroneand similar water insoluble steroidal drugs. One or more compounds ofthe invention (herein referred to as the active ingredients) areadministered by any route appropriate to the condition to be treated.Suitable topical routes include oral, rectal, nasal, topical (includingbuccal and sublingual), and vaginal, preferably across the epidermis.Although not preferred, the compositions of the invention can also beused for parenteral administration (including subcutaneous,intramuscular, intradermal, intrathecal and epidural), and the like. Itwill be appreciated that the preferred route may vary with for examplethe condition of the recipient. An advantage of the compositions of thisinvention is that they can be topically administered.

Some of the examples of methods for formulating transdermal compositionsthat may be useful for formulating compositions herein are disclosed inU.S. Pat. Nos. 7,198,801 and 7,244,703. Cyclopentadecalactone andcyclohexadecanone may be useful as additives in formulating transdermalcompositions herein.

Pretreatment of C3H/HeN mice with s.c. DHEA (60 mg/kg) for 3 dayssignificantly reduces dexamethasone (DEX)-induced thymic and splenicatrophy one day after a single DEX dose (1.6 mg) (Blauer et. al.,Endocrinology 129:3174-3179, 1991). Using this model, we confirmed thatDHEA at a dose of 60 mg/kg significantly protected CD-1 mice againstDEX-induced thymic and splenic atrophy, but was inactive at a dose of 10mg/kg or 20 mg/kg. In contrast, fluasterone at a dose of 10 mg/kg was asactive as DHEA at 60 mg/kg and also showed significant protection at adose of 5 mg/kg.

In addition to fluasterone and DHEA, nine other related steroids weretested for efficacy in protecting mice against DEX-induced thymic andsplenic involution. Fluasterone was the most active steroid tested, withthe steroid 3β-methyl-5-androsten-17-one showing almost as greatpotency. The other steroids tested were all significantly less activethan fluasterone. The various steroids tested, as well as their potencyas compared to fluasterone, are shown in the following table.

TABLE 1 Relative potency against Dexamethasone induced thymic andsplenic atrophy. Approximate Potency Compared to Fluasterone inProtecting Against DEX- induced Thymic and Steroid Splenic InvolutionFluasterone 1 DHEA about ⅙ 16α-fluoro-5α-androstan-17-one (8356) about ⅛7α-hydroxyfluasterone about ½ 16α-methyl-5-androsten-17-one about ⅙ orless 16α-hydroxy-5-androsten-17-one about 1/1216α-fluoro-5-androsten-17β-ol < 1/12 3β-methyl-5-androsten-17-oneslightly less active 3β-methyl-16α-fluoro-5-androsten-17-one <½3β-methyl-16α-chloro-5-androsten-17-one about ⅙ or considerably less3β-methyl-16α-methyl-5-androsten-17-one about ⅙

The compositions of the present invention are also useful for thetreatment or prevention of diabetes as well as treatment and preventionof obesity. Without being bound by any theory, the observations hereinindicate that the anti-glucocorticoid action of these steroids may beresponsible for their anti-obesity activity. Very likely, theanti-glucocorticoid action is the primary mechanism of the anti-diabeticeffect as well.

The compositions of the present invention are useful for the treatment,control, or prevention of obesity-related disorders. The obesity-relateddisorders herein are associated with, caused by, or result from obesity.Examples of obesity-related disorders include diabetes, overeating,binge eating, and bulimia, hypertension, elevated plasma insulinconcentrations and insulin resistance, dyslipidemias, hyperlipidemia,endometrial, breast, prostate, kidney and colon cancer, osteoarthritis,obstructive sleep apnea, gallstones, heart disease, abnormal heartrhythms and arrythmias, myocardial infarction, congestive heart failure,coronary heart disease, sudden death, stroke, polycystic ovary disease,craniopharyngioma, atherosclerosis, the Prader-Willi Syndrome,Frohlich's syndrome, GH-deficient subjects, normal variant shortstature, Turner's syndrome, and other pathological conditions showingreduced metabolic activity or a decrease in resting energy expenditureas a percentage of total fat-free mass, e.g, children with acutelymphoblastic leukemia. Further examples of obesity-related disordersare metabolic syndrome, also known as syndrome X, insulin resistancesyndrome, reproductive hormone abnormalities, sexual and reproductivedysfunction, such as impaired fertility, infertility, hypogonadism inmales and hirsutism in females, fetal defects associated with maternalobesity, gastrointestinal motility disorders, such as obesity-relatedgastro-esophageal reflux, breathlessness, respiratory disorders, such asobesity-hypoventilation syndrome (Pickwickian syndrome), cardiovasculardisorders, inflammation, such as systemic inflammation of thevasculature, atheriosclerosis, hypercholesterolemia, hyperuricaemia,lower back pain, gallbladder disease, hyperuricemia, gout, and kidneycancer, and increased anesthetic risk. The compositions of the presentinvention are also useful to treat Alzheimer's disease.

Dyslipidemias or disorders of lipid metabolism, include variousconditions characterized by abnormal concentrations of one or morelipids (i.e. cholesterol and triglycerides), and/or apolipoproteins(i.e., apolipoproteins A, B, C and E), and/or lipoproteins (i.e., themacromolecular complexes formed by the lipid and the apolipoprotein thatallow lipids to circulate in blood, such as LDL, VLDL and IDL).Hyperlipidemia is associated with abnormally high levels of lipids, LDLand VLDL cholesterol, and/or triglycerides. Treatment of dyslipidemiarefers to the administration of the combinations of the presentinvention to a dyslipidemic subject. Prevention of dyslipidemia refersto the administration of the combinations of the present invention to apre-dyslipidemic subject. A pre-dyslipidemic subject is a subject withhigher than normal lipid levels, that is not yet dyslipidemic.

The term “metabolic syndrome”, also known as syndrome X, is defined inthe Third Report of the National Cholesterol Education Program ExpertPanel on Detection, Evaluation and Treatment of High Blood Cholesterolin Adults (ATP-III). E. S. Ford et al., Journal of the American MedicalAssociation, vol. 287 (3), Jan. 16, 2002, pp 356-359. Briefly, a personis defined as having metabolic syndrome if the person has three or moreof the following disorders: abdominal obesity, hypertriglyceridemia, lowHDL cholesterol, high blood pressure, and high fasting plasma glucose.The criteria for these are defined in ATP-m. Treatment of metabolicsyndrome refers to the administration of the combinations of the presentinvention to a subject with metabolic syndrome. Prevention of metabolicsyndrome refers to the administration of the combinations of the presentinvention to a subject with two of the disorders that define metabolicsyndrome. A subject with two of the disorders that define metabolicsyndrome is a subject that has developed two of the disorders thatdefine metabolic syndrome, but has not yet developed three or more ofthe disorders that define metabolic syndrome.

The term “obesity” as used herein is a condition in which there is anexcess of body fat. The operational definition of obesity is based onthe Body Mass Index (BMI), which is calculated as body weight per heightin meters squared (kg/m²). “Obesity” refers to a condition whereby anotherwise healthy subject has a Body Mass Index (BMI) greater than orequal to 30 kg/m² or a condition whereby a subject with at least oneco-morbidity has a BMI greater than or equal to 27 kg/m.sup.2. An “obesesubject” is an otherwise healthy subject with a Body Mass Index (BMI)greater than or equal to 30 kg/m² or a subject with at least oneco-morbidity with a BMI greater than or equal to 27 kg/m². A “subject atrisk of obesity” is an otherwise healthy subject with a BMI of 25 kg/m²to less than 30 kg/m² or a subject with at least one co-morbidity with aBMI of 25 kg/m² to less than 27 kg/m².

The increased risks associated with obesity occur at a lower Body MassIndex (BMI) in Asians. In Asian countries, including Japan, “obesity”refers to a condition whereby a subject with at least oneobesity-induced or obesity-related co-morbidity, that requires weightreduction or that would be improved by weight reduction, has a BMIgreater than or equal to 25 kg/m². In Asian countries, including Japan,an “obese subject” refers to a subject with at least one obesity-inducedor obesity-related co-morbidity that requires weight reduction or thatwould be improved by weight reduction, with a BMI greater than or equalto 25 kg/m². In Asia-Pacific, a “subject at risk of obesity” is asubject with a BMI of greater than 23 kg/m² to less than 25 kg/m².

Obesity-induced or obesity-related co-morbidities include, but are notlimited to, diabetes, non-insulin dependent diabetes mellitus—type 2,diabetes associated with obesity, impaired glucose tolerance, impairedfasting glucose, insulin resistance syndrome, dyslipidemia,hypertension, hypertension associated with obesity, hyperuricacidemia,gout, coronary artery disease, myocardial infarction, angina pectoris,sleep apnea syndrome, Pickwickian syndrome, fatty liver; cerebralinfarction, cerebral thrombosis, transient ischemic attack, orthopedicdisorders, arthritis deformans, lumbodynia, emmeniopathy, andinfertility. In particular, co-morbidities include: hypertension,hyperlipidemia, dyslipidemia, glucose intolerance, cardiovasculardisease, sleep apnea, diabetes mellitus, and other obesity-relatedconditions.

Treatment of obesity and obesity-related disorders refers to theadministration of the compounds or combinations of the present inventionto reduce or maintain the body weight of an obese subject. One outcomeof treatment may be reducing the body weight of an obese subjectrelative to that subject's body weight immediately before theadministration of the compounds or combinations of the presentinvention. Another outcome of treatment may be preventing body weightregain of body weight previously lost as a result of diet, exercise, orpharmacotherapy. Another outcome of treatment may be decreasing theoccurrence of and/or the severity of obesity-related diseases. Thetreatment may suitably result in a reduction in food or calorie intakeby the subject, including a reduction in total food intake, or areduction of intake of specific components of the diet such ascarbohydrates or fats; and/or the inhibition of nutrient absorption;and/or the inhibition of the reduction of metabolic rate; and in weightreduction in patients in need thereof. The treatment may also result inan alteration of metabolic rate, such as an increase in metabolic rate,rather than or in addition to an inhibition of the reduction ofmetabolic rate; and/or in minimization of the metabolic resistance thatnormally results from weight loss.

Prevention of obesity and obesity-related disorders refers to theadministration of the compounds or combinations of the present inventionto reduce or maintain the body weight of a subject at risk of obesity.One outcome of prevention may be reducing the body weight of a subjectat risk of obesity relative to that subject's body weight immediatelybefore the administration of the compounds or combinations of thepresent invention. Another outcome of prevention may be preventing bodyweight regain of body weight previously lost as a result of diet,exercise, or pharmacotherapy. Another outcome of prevention may bepreventing obesity from occurring if the treatment is administered priorto the onset of obesity in a subject at risk of obesity. Another outcomeof prevention may be decreasing the occurrence and/or severity ofobesity-related disorders if the treatment is administered prior to theonset of obesity in a subject at risk of obesity. Moreover, if treatmentis commenced in already obese subjects, such treatment may prevent theoccurrence, progression or severity of obesity-related disorders, suchas, but not limited to, arteriosclerosis, Type 2 diabetes, polycysticovary disease, cardiovascular diseases, osteoarthritis, dermatologicaldisorders, hypertension, insulin resistance, hypercholesterolemia, andhypertriglyceridemia.

Topical applications of the instant application are also useful for thetreatment of osteoarthritis of the joints. In particular the hand, foot,thumb, forearm, knees, hip, jaw and elbow joints can be treated locallywith topical applications containing compounds of formula I and II.Metacarpophalangeal, temporomandibular, trapeziometacarpal, metatarsal,carpus, ginglymus and sternoclavicular articulation joints can betreated with compositions of the instant application.

Formulations of Poorly Water Soluble Compounds:

The applicants have surprisingly found that certain mixtures ofsurfactants and/or organic alcohols can significantly improve thesolubility of compounds of formula I. Such improved solubility is usefulfor preparing pharmaceutical formulations containing compounds offormula I and TI for topical, oral and subcutaneous administration.Preferable surfactants include polysorbates, and long-chain organicalcohols, including cetyl alcohol, stearyl alcohol etc. Polysorbate isused as the preferred surfactant, with Polysorbate 80 (polyoxyethylene(20) sorbitan monooleate; Tween 80) being an especially preferredsurfactant. Sorbitan monooleate or other polysorbates with varyingpolyoxyethylene chain lengths can also be used. Straight chain organicalcohols with chain lengths in the range of 8-30 carbons are also usefulas surfactants. Especially preferred carbon chain length is in the rangeof 14-24. Such compounds can correspond to the formula CH₃(CH₂)_(n)—OH,wherein n is 13-23. Another group of compounds useful as surfactants ispolyethyleneglycol conjugated fatty acids and alcohols. Particularlypreferred in this group of surfactants are the polyethyleneglycolstearate (MYRJ™ 45), Macrogol stearyl ether 2 (BRIJ™ 72), Macrogolstearyl ether 20 (BRIJ™ 72P), Macrogol stearyl ether 20-23 (BRIJ™ 35P),Macrogol stearate 40-50 (MYRJ™ 52S), Macrogol stearate 100 (MYRJ™ 25P),Macrogolglycerol hydroxystearate 25 (ATLAS™), Macrogolglycerol laurylether 9 and Macrogolglycerol lauryl ether 9. Sorbitan substituted fattyacids are also useful as surfactants in formulations of Formula I andII. For example, Sorbitan laureate, Sorbitan stearate, Sorbitan oleateand Sorbitan trioleate can be used.

In one embodiment, the formulations herein can be in the form of aqueousgel, anhydrous gel, a water-in-oil emulsion, oil-in-water emulsion or asuspension. Examples of gel forming procedure for DHEA can be found inU.S. Pat. Nos. 5,709,878, and 4,978,532 the entire content of which areincorporated by reference herein. Gels are semisolid systems of eithercontaining suspended small inorganic particles (two phase gels) ororganic macromolecules interpenetrated by a liquid (single phase gels).Emollients such as petrolatum, paraffin wax, beeswax, cetyl palmitate,and lanolin can be included in the formulations herein. When formulatedfor presentation as a gel, the composition of the invention can includea gelling agent such as a finely divided solid and/or a thickener inconcentrations that produce a loose molecular network inhibiting thefree movement of liquid ingredients. Thus a typical gel composition ofthe invention includes a concentration of a compound of Formula I or IIin the range of about 0.1 to about 20 grams per 100 grams ofcomposition, preferably about 0.25 to about 5 grams per 100 grams; aconcentration of phospholipid in the range of about 2 to about 50 gramsper 100 grams of composition, preferably about 3 to about 25 grams per100 milliliters; a concentration of finely divided solid in the range ofabout 0 to about 15 grams per 100 grams of composition, and aconcentration of thickener in the range of about 0 to about 15 grams per100 grams of composition.

Gellants may also be included in the formulations. These agents aretypically non-ionic or cationic polymers such as hydroxyethyl cellulose,methylcellulose, guar gum, xanthan gum, hydroxypropylcellulose andcationic cellulosics. A particular example is Sepigel.

In one embodiment, a gel comprising a compound of formula I or II, canbe made by mixing a lower alkyl alcohol, a polysorbate, water and acompound of formula I or II and, optionally, adding and mixing athickening agent followed by incubating the ingredients until gelformation. Various temperatures may be used for incubation to effect gelformation. A preferred temperature range is about 3° C. to about 90° C.;a more preferred range is about 10° C. to about 50° C.; and morepreferred range is about 10° C. to about 40° C. Incubation times varydepending on the temperature, and the ratio of ingredients. The ratiosof ingredients may also vary depending on the particular compound offormula I or II and the particular lower alcohol use. The compositionmay comprise alcohol in the range of from about 20 to about 95% (v/v);preferably from about 30 to about 90%; even more preferably about 50 toabout 90%. The water content may from about 0 to about 60%; preferablyabout 2 to about 40%; more preferably about 5 to about 30%; even morepreferably about 15 to about 30%. The surfactant may be present in therange of about 0 to 10%; more preferably about 0.01% to about 5%; evenmore preferably about 0.01% to about 3.5%.

Examples of thickening agents that can be added to the gel or solutionformulations described herein include: cellulosic thickening agents, forexample, cellulose, hydroxyethyl-cellulose, carboxymethylcellulose, andhydroxypropylmethyl-cellulose; and acrylic thickening agents. Examplesof preferred acrylic thickeners are carbomers, for example, non-linearpolymers of acrylic acid cross-linked with a polyalkenyl polyether.Examples of preferred carbomers which may be used in the presentinvention include carboxypolymethylene, carboxyvinyl polymer, and alkylacrylates, for example, acrylic acid/alkyl methacrylate copolymer. Allof the above are available from Noveon, with carboxypolymethylene soldas Carbopol 980®, carboxyvinyl polymer sold as Carbopol 940®, andacrylic acid/alkyl methacrylate copolymer sold as Pemulen TR-1®.

In a preferred embodiment, the formulations of the invention can beapplied by misting or spraying the formulation on the skin either via ametered dose device or from a unit dose container. In this method, theformulation can be distributed evenly over a larger area therebyproviding a quick means for absorption. Alternatively the formulationcan be applied via an applicator, such as a roll-on applicator, ametered pump dispenser or sponge.

In one embodiment, the composition of this invention is administered tothe recipient by means of a transdermal delivery system or patch.Transdermal delivery is accomplished by exposing a source of thesubstance to be administered to the recipient's skin for an extendedperiod of time. Typically, the formulation is incorporated in orabsorbed on a matrix or container from which it is released onto therecipient's skin. The rate of release can be controlled by a membraneplaced between the container and the skin, by diffusion directly fromthe container, or by the skin itself serving as a rate-controllingbarrier. Many suitable transdermal delivery systems and containerstherefore are known, ranging in complexity from a simple gauze padimpregnated with the substance to be administered and secured to theskin with an adhesive bandage to multilayer and multi-componentstructures. Some of the systems are characterized by the use with thesubstance to be administered of a shaped article sufficiently flexibleto snugly fit to the skin of the recipient and thus serve both ascontainer from which the substance is delivered to the recipient's skinand as barrier to prevent loss or leakage of the substance away from thearea of the skin to which the substance is to be delivered. Atransdermal delivery system or patch may also contain an added substancethat assists the penetration of the active ingredient through the skin,usually termed a skin enhancer or penetration enhancer. Transdermaldelivery systems may contain an ethoxylated oil such as ethoxylatedcastor oil, ethoxylated jojoba oil, ethoxylated corn oil, andethoxylated emu oil. An alcohol mixed with the ethoxylated oil may forma penetration enhancer.

A topical oil-in-water emulsion composition can be prepared by making asolution of fluasterone (or related compound) as described above andadding an immiscible phase (e.g., a biocompatible oil phase) and anoptional emulsifying agent. An irritation mitigating agent can also beincluded, such as C₁₂₋₁₅ alkyl benzoate, octyl methoxycinnamate, octyldimethyl PABA, octocrylene, menthyl anthranilate, and homomenthylsalicylate.

In certain preferred embodiments a foam comprising compounds of instantapplication can be prepared. An example of a foam forming procedure canbe found in U.S. Pat. No. 7,141,237. For instance, an active agent in asolution as described herein and a quick-breaking foaming agentcomprising a mixture of cetyl alcohol and stearyl alcohol, which aredissolved in the ethanol solution can be used. Preferably, thiscomposition is packaged in a polyamide-imide-lined aluminum can andpressurized with a propane/butane mixture as the propellant. Under thepackaged pressure, the hydrocarbon propellant liquefies and becomesmiscible with the water/ethanol solution.

The formulation herein may contain an emulsifier and/or surfactant. Awide variety of such agents can be employed. In one embodiment, thecompositions of the present invention comprise from about 0.05% to about95%, preferably from about 10% to about 80%, and more preferably fromabout 3.5% to about 60% of at least one surfactant. The surfactant, at aminimum, must be hydrophilic enough to disperse in ethanol or othersolvent system. The surfactants useful herein can include any of a widevariety of cationic, anionic, zwitterionic, and amphoteric surfactantsdisclosed in prior patents and other references. The exact surfactantchosen will depend upon the pH of the composition and the othercomponents present.

In one embodiment, the composition comprises a hydrophilic emulsifier orsurfactant. The compositions of the present invention preferablycomprise from about 0.05% to about 5%, more preferably from about 0.05%to about 3.5% of at least one hydrophilic surfactant. Without intendingto be limited by theory, it is believed that the hydrophilic surfactantassists in dispersing hydrophobic materials.

Preferred hydrophilic surfactants are selected from nonionicsurfactants. Among the nonionic surfactants that are useful herein arethose that can be broadly defined as condensation products of long chainalcohols, e.g. C8-30 alcohols, with sugar or starch polymers, i.e.,glycosides. These compounds can be represented by the formula(S)_(n)—O—R wherein S is a sugar moiety such as glucose, fructose,mannose, and galactose; n is an integer of from about 1 to about 1000,and R is a C8-30 alkyl group. Examples of long chain alcohols from whichthe alkyl group can be derived include decyl alcohol, cetyl alcohol,stearyl alcohol, lauryl alcohol, myristyl alcohol, oleyl alcohol, andthe like. Preferred examples of these surfactants include those whereinS is a glucose moiety, R is a C8-20 alkyl group, and n is an integer offrom about 1 to about 9. Commercially available examples of thesesurfactants include decyl polyglucoside and lauryl polyglucoside.

Other useful nonionic surfactants include the condensation products ofalkylene oxides with fatty acids (i.e. alkylene oxide esters of fattyacids); the condensation products of alkylene oxides with 2 moles offatty acids (i.e. alkylene oxide diesters of fatty acids); thecondensation products of alkylene oxides with fatty alcohols (i.e.alkylene oxide ethers of fatty alcohols); and the condensation productsof alkylene oxides with both fatty acids and fatty alcohols. Nonlimitingexamples of these alkylene oxide derived nonionic surfactants includeceteth-6, ceteth-10, ceteth-12, ceteareth-6, ceteareth-10, ceteareth-12,steareth-6, steareth-10, steareth-12, PEG-6 stearate, PEG-10 stearate,PEG-100 stearate, PEG-12 stearate, PEG-20 glyceryl stearate, PEG-80glyceryl tallowate, PEG-10 glyceryl stearate, PEG-30 glyceryl cocoate,PEG-80 glyceryl cocoate, PEG-200 glyceryl tallowate, PEG-8 dilaurate,PEG-10 distearate, and mixtures thereof.

Other nonionic surfactants suitable for use herein include sugar estersand polyesters, alkoxylated sugar esters and polyesters, C1-C30 fattyacid esters of C1-C30 fatty alcohols, alkoxylated derivatives of C1-C30fatty acid esters of C1-C30 fatty alcohols, alkoxylated ethers of C1-C30fatty alcohols, polyglyceryl esters of C1-C30 fatty acids, C1-C30 estersof polyols, C1-C30 ethers of polyols, alkyl phosphates, polyoxyalkylenefatty ether phosphates, fatty acid amides, acyl lactylates, and mixturesthereof. Nonlimiting examples of these non-silicon-containingemulsifiers include: polyethylene glycol 20 sorbitan monolaurate(Polysorbate 20), polyethylene glycol 5 soya sterol, Steareth-20,Ceteareth-20, PPG-2 methyl glucose ether distearate, Ceteth-10,Polysorbate 80, cetyl phosphate, potassium cetyl phosphate,diethanolamine cetyl phosphate, Polysorbate 60, glyceryl stearate,polyoxyethylene 20 sorbitan trioleate (Polysorbate 85), sorbitanmonolaurate, polyoxyethylene 4 lauryl ether sodium stearate,polyglyceryl-4 isostearate, hexyl laurate, PPG-2 methyl glucose etherdistearate, PEG-100 stearate, and mixtures thereof. Commerciallyavailable surfactants include polysorbate 80 (Tween 80), polysorbate 20(Tween 20), polysorbate 40 (Tween 40) and polysorbate (60). Thepreferred surfactants include polysorbates and more preferred surfactantis Tween 80.

Other emulsifiers useful herein are fatty acid ester blends based on amixture of sorbitan or sorbitol fatty acid ester and sucrose fatty acidester, the fatty acid in each instance being preferably C8-C24, morepreferably C10-C20. The preferred fatty acid ester emulsifier is a blendof sorbitan or sorbitol C16-C20 fatty acid ester with sucrose C10-C16fatty acid ester, especially sorbitan stearate and sucrose cocoate.

The hydrophilic surfactants useful herein can alternatively oradditionally include any of a wide variety of cationic, anionic,zwitterionic, and amphoteric surfactants known in the art. The cationicsurfactants useful herein include cationic ammonium salts such asquaternary ammonium salts, and amino-amides.

A wide variety of anionic surfactants are also useful herein.Nonlimiting examples of anionic surfactants include the alkoylisethionates (e.g., C12-C30), alkyl and alkyl ether sulfates and saltsthereof, alkyl and alkyl ether phosphates and salts thereof, alkylmethyl taurates (e.g., C12-C30), and soaps (e.g., alkali metal salts,e.g., sodium or potassium salts) of fatty acids.

Amphoteric and zwitterionic surfactants are also useful herein. Examplesof amphoteric and zwitterionic surfactants which can be used in thecompositions of the present invention are those which are broadlydescribed as derivatives of aliphatic secondary and tertiary amines inwhich the aliphatic radical can be straight or branched chain andwherein one of the aliphatic substituents contains from about 8 to about22 carbon atoms (preferably C8-C 18) and one contains an anionic watersolubilizing group, e.g., carboxy, sulfonate, sulfate, phosphate, orphosphonate. Examples are alkyl imino acetates, and iminodialkanoatesand aminoalkanoates, imidazolinium and ammonium derivatives. Othersuitable amphoteric and zwitterionic surfactants are those selected fromthe group consisting of betaines, sultaines, hydroxysultaines, alkylsarcosinates (e.g., C12-C30), and alkanoyl sarcosinates.

The compositions hereof, and especially the emulsions hereof, maycontain a structuring agent. Structuring agents are particularlypreferred in the oil-in-water emulsions of the present invention.Without being limited by theory, it is believed that the structuringagent assists in providing rheological characteristics to thecomposition which contribute to the stability of the composition. Forexample, the structuring agent tends to assist in the formation of theliquid crystalline gel network structures. The structuring agent mayalso function as an emulsifier or surfactant. Preferred compositions ofthis invention comprise from about 1% to about 90%, more preferably fromabout 1% to about 60% of one or more structuring agents.

Suitable structuring agents of the present invention are selected fromthe group consisting of palmitic acid, stearyl alcohol, cetyl alcohol,behenyl alcohol, stearic acid, palmitic acid, the polyethylene glycolether of stearyl alcohol having an average of about 1 to about 5ethylene oxide units, the polyethylene glycol ether of cetyl alcoholhaving an average of about 1 to about 5 ethylene oxide units, andmixtures thereof. More preferred structuring agents of the presentinvention are selected from the group consisting of stearyl alcohol,cetyl alcohol, behenyl alcohol, the polyethylene glycol ether of stearylalcohol having an average of about 2 ethylene oxide units (steareth-2),the polyethylene glycol ether of cetyl alcohol having an average ofabout 2 ethylene oxide units, and mixtures thereof. Even more preferredstructuring agents are selected from the group consisting of stearicacid, palmitic acid, stearyl alcohol, cetyl alcohol, behenyl alcohol,steareth-2, and mixtures thereof.

Another aspect of the invention herein is the use of nanosized compoundsof formula I and II, in particular fluasterone, for topicaladministration. Because of the high adhesiveness of nanoparticles onbiological surfaces (e.g., epithelial gut wall), nanoparticulatetechnology may prolong the absorption time of poorly soluble drugs,thereby improving bioavailability. There are several well known methodsfor the preparation of nanosized pharmaceuticals. For example, wetmilling or piston gap homogenization can be used to nanosizefluasterone. For discussions related to wet milling, see, e.g., U.S.Pat. No. 5,518,187 to J. A. Bruno et al.; U.S. Pat. No. 5,862,999 (D. A.Czekai and L. P. Seaman); and U.S. Pat. No. 5,534,270 (L. De Castro);for discussions related to piston gap homogenization, see U.S. Pat. No.5,543,133 (J. R. Swanson et al.); U.S. Pat. No. 5,858,410 (R. H. Mulleret al.); U.S. Patent Publication No. 2003/0072807 A1 (J. C-T. Wong etal.); and U.S. Pat. No. 5,510,118 (H. W. Bosch et al.), the completedisclosures of which are herein incorporated by reference. Wet millingis a well understood process, which relies on impact and shear forces toreduce particle size. Piston gap homogenization, which utilizescavitation forces and impact or shear forces to reduce particle size. Amethod involving high-pressure spray homogenizer can also be used toprepare nanosized particles. (Galli et. al. U.S. Patent Publication No.20070020197).

The nanosized or micronized compounds of formula I and II can be part ofa delivery matrix. For example, matrix carrier can be an amorphousmicroporous non-fibrous silicon or titanium oxide similar to thosedescribed in U.S. Patent Publication No. 20070275068 can be used.Sol-gel processed drug-silica composite materials have been investigatedfor controlled drug release. One concept involving the use of sol-geltype silica is the synthesis of a bio-erodible silica-drug composite.Silica-based drug release systems prepared using sol-gel approaches inwhich compounds of formulae I and II are introduced duringpolymerisation and processing of the silica matrix can be used for drugdelivery. An alternative approach for making a drug delivery systembased on silica gels is the synthesis of silica in the absence ofcompounds of formulae I and II, followed by drying and calcination toobtain a xerogel and then by loading the calcinated material with theappropriate compound. The sol-gel approach enables the synthesis of alarge variety of silica materials. Micropores with very narrow pore sizedistribution can be obtained through calcinations which can be usefulfor the delivery of micronized or nano-sized compounds of formulae I andII, particularly for topical delivery.

The term “atherosclerosis” as used herein encompasses vascular diseasesand conditions that are recognized and understood by physicianspracticing in the relevant fields of medicine. Atheroscleroticcardiovascular disease, coronary heart disease (also known as coronaryartery disease or ischemic heart disease), cerebrovascular disease andperipheral vessel disease are all clinical manifestations ofatherosclerosis and are therefore encompassed by the terms“atherosclerosis” and “atherosclerotic disease.” The combinationcomprised of a therapeutically effective amount of an anti-obesity agentin combination with a therapeutically effective amount of ananti-hypertensive agent may be administered to prevent or reduce therisk of occurrence, or recurrence where the potential exists, of acoronary heart disease event, a cerebrovascular event, or intermittentclaudication. Coronary heart disease events are intended to include CHDdeath, myocardial infarction (i.e., a heart attack), and coronaryrevascularization procedures. Cerebrovascular events are intended toinclude ischemic or hemorrhagic stroke (also known as cerebrovascularaccidents) and transient ischemic attacks. Intermittent claudication isa clinical manifestation of peripheral vessel disease. The term“atherosclerotic disease event” as used herein is intended to encompasscoronary heart disease events, cerebrovascular events, and intermittentclaudication. It is intended that persons who have previouslyexperienced one or more non-fatal atherosclerotic disease events arethose for whom the potential for recurrence of such an event exists.

The term “diabetes,” as used herein, includes both insulin-dependentdiabetes mellitus (i.e., IDDM, also known as type 1 diabetes) andnon-insulin-dependent diabetes mellitus (i.e., NIDDM, also known as Type2 diabetes). Type 1 diabetes, or insulin-dependent diabetes, is theresult of an absolute deficiency of insulin, the hormone which regulatesglucose utilization. Type 2 diabetes, or insulin-independent diabetes(i.e., non-insulin-dependent diabetes mellitus), often occurs in theface of normal, or even elevated levels of insulin and appears to be theresult of the inability of tissues to respond appropriately to insulin.Most of the Type 2 diabetics are also obese. The compositions of thepresent invention are useful for treating both Type 1 and Type 2diabetes. The term “diabetes associated with obesity” refers to diabetescaused by obesity or resulting from obesity. The compositions areespecially effective for treating Type 2 diabetes. The compositions ofthe present invention are also useful for treating and/or preventinggestational diabetes mellitus.

Diabetes is characterized by a fasting plasma glucose level of greaterthan or equal to about 126 mg/dl. A diabetic subject has a fastingplasma glucose level of greater than or equal to about 126 mg/dl.Prediabetes is characterized by an impaired fasting plasma glucose (FPG)level of greater than or equal to about 110 mg/dl and less than about126 mg/dl; or impaired glucose tolerance; or insulin resistance. Aprediabetic subject is a subject with impaired fasting glucose (afasting plasma glucose (FPG) level of greater than or equal to about 110mg/dl and less than about 126 mg/dl); or impaired glucose tolerance; orinsulin resistance, resulting in an increased risk of developingdiabetes.

Treatment of diabetes mellitus refers to the administration of acompound or combination of the present invention to treat a diabeticsubject. One outcome of treatment may be decreasing the glucose level ina subject with elevated glucose levels. Another outcome of treatment maybe decreasing insulin levels in a subject with elevated insulin levels.Another outcome of treatment may be decreasing plasma triglycerides in asubject with elevated plasma triglycerides. Another outcome of treatmentis decreasing LDL cholesterol in a subject with high LDL cholesterollevels. Another outcome of treatment may be increasing HDL cholesterolin a subject with low HDL cholesterol levels. Another outcome oftreatment is increasing insulin sensitivity. Another outcome oftreatment may be enhancing glucose tolerance in a subject with glucoseintolerance. Yet another outcome of treatment may be decreasing insulinresistance in a subject with increased insulin resistance or elevatedlevels of insulin. Prevention of diabetes mellitus, in particulardiabetes associated with obesity, refers to the administration of acompound or combination of the present invention to prevent the onset ofdiabetes in a subject in need thereof. A subject in need of preventingdiabetes is a prediabetic subject that is overweight or obese.

The terms “administration of” and or “administering a” compound shouldbe understood to mean providing a compound of the invention or a prodrugof a compound of the invention to a subject in need of treatment. Theinstant pharmaceutical compositions include administration of a singlepharmaceutical dosage formulation which contains the anti-obesity agentand an anti-hypertensive agent, for example, as well as administrationof each active agent in its own separate pharmaceutical dosageformulation. Where separate dosage formulations are used, the individualcomponents of the composition can be administered at essentially thesame time, i.e., concurrently, or at separately staggered times, i.e.sequentially prior to or subsequent to the administration of the othercomponent of the composition. The instant pharmaceutical composition istherefore to be understood to include all such regimes of simultaneousor alternating treatment, and the terms “administration” and“administering” are to be interpreted accordingly. Administration inthese various ways are suitable for the present compositions as long asthe beneficial pharmaceutical effect of the combination of theanti-obesity agent and the anti-hypertensive agent is realized by thepatient at substantially the same time. Such beneficial effect ispreferably achieved when the target blood level concentrations of eachactive drug are maintained at substantially the same time. A singledosage formulation will provide convenience for the patient, which is animportant consideration especially for patients with diabetes, metabolicsyndrome, or obese patients who may be in need of multiple medications.

The term “subject”, as used herein refers to an animal, preferably amammal, most preferably a human, who has been the object of treatment,observation or experiment. In one embodiment the term “mammal” is a“human” said human being either male or female. The instant combinationsare also useful for treating or preventing obesity and obesity-relateddisorders in cats and dogs. As such, the term “mammal” includescompanion animals such as cats and dogs.

The term “subject in need thereof” refers to a subject who is in need oftreatment or prophylaxis as determined by a researcher, veterinarian,medical doctor or other clinician. In one embodiment a subject in needthereof is a mammal. In another embodiment, a subject in need thereof isan obese and/or diabetic subject or a subject who is at risk of becomingdiabetic.

The administration of the composition of the present invention in orderto practice the present methods of therapy is carried out byadministering a therapeutically effective amount of the compounds in thecomposition to a subject in need of such treatment or prophylaxis. Theneed for a prophylactic administration according to the methods of thepresent invention is determined via the use of well known risk factors.The effective amount of an individual compound is determined, in thefinal analysis, by the physician in charge of the case, but depends onfactors such as the exact disease to be treated, the severity of thedisease and other diseases or conditions from which the patient suffers,the chosen route of administration, other drugs and treatments which thepatient may concomitantly require, and other factors in the physician'sjudgment.

The term “therapeutically effective amount” as used herein means theamount of the active compounds in the composition that will elicit thebiological or medical response in a tissue, system, subject, or humanthat is being sought by the researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisorder being treated. The novel methods of treatment of this inventionare for disorders known to those skilled in the art.

The term “prophylactically effective amount” as used herein means theamount of the active compounds in the composition that will elicit thebiological or medical response in a tissue, system, subject, or humanthat is being sought by the researcher, veterinarian, medical doctor orother clinician, to prevent the onset of diabetes, for example.

The magnitude of prophylactic or therapeutic dose of the activeingredients of the composition will, of course, vary with the nature ofthe severity of the condition to be treated and with the particularcompound in the composition and its route of administration. It willalso vary according to the age, weight and response of the individualpatient In general, for treating, controlling, and/or preventingmetabolic syndrome, the anti-hypertensive agent, the anti-obesity agent,the anti-diabetic agent and the anti-dyslipidemic agent in thecombination are administered at a daily dosage of from about 0.0001mg/kg to about 1000 mg/kg of body weight, preferably from about 0.001mg/kg to about 100 mg/kg, given in a single dose or in divided doses twoto six times per day, or in sustained release form. The dosage regimenmay be adjusted to provide the optimal therapeutic response.

The compounds of this invention can be administered to humans in thedosage ranges specific for each compound. The effective dosage of eachof the active ingredients employed in the composition may vary dependingon the particular compound employed, the mode of administration, thecondition being treated and the severity of the condition being treated.Thus, the dosage regimen utilizing the compositions of the presentinvention is selected in accordance with a variety of factors includingtype, species, age, general health, body weight, diet, sex and medicalcondition of the subject; the severity of the condition to be treated;the renal and hepatic function of the patient; the drug combination; andthe particular compounds employed and their routes of administration. Aphysician, clinician or veterinarian of ordinary skill can readilydetermine and prescribe the effective amount of the drug required toprevent, counter or arrest the progress of the condition.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. All patents, patentapplications, published applications and publications, websites andother published materials referred to throughout the entire disclosureherein, unless noted otherwise, are incorporated by reference in theirentirety. In the event that there are a plurality of definitions forterms herein, those in this section prevail. Reference thereto evidencesthe availability and public dissemination of such information. It is tobe understood that both the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of the products, methods and other subject matterprovided herein. In this application, the use of the singular includesthe plural unless specifically stated otherwise. In this application,the use of “or” means “and/or” unless stated otherwise. Furthermore, useof the term “including” as well as other forms, such as “includes,” and“included,” is not limiting.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in the applicationincluding, but not limited to, patents, patent applications, articles,books, manuals, and treatises are hereby expressly incorporated byreference in their entirety for any purpose.

Unless specific definitions are provided, the nomenclatures utilized inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those known in the art.Standard techniques can be used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

As used herein, the term “alkyl” refers to an aliphatic hydrocarbongroup. An alkyl group can be a “saturated alkyl,” which means that itdoes not contain any alkene or alkyne groups. An alkyl group can be an“unsaturated alkyl,” which means that it contains at least one alkene oralkyne group. An alkyl, whether saturated or unsaturated, can bebranched, straight chain, or cyclic.

As used herein, the term “lower alkyl” refers to an alkyl containing 1to 5 carbon atoms. The term “medium alkyl” refers to an alkyl containing5 to 10 carbon atoms. An alkyl can be designated as “C₁-C₄ alkyl” orsimilar designations. By way of example only, “C₁-C₄ alkyl” indicates analkyl having one, two, three, or four carbon atoms, i.e., the alkyl isselected from among methyl, ethyl, propyl, iso-propyl, n-butyl,iso-butyl, sec-butyl, and t-butyl. Thus C₁-C₄ includes C₁-C₂ and C₂-C₃alkyl. Alkyls can be substituted or unsubstituted. Alkyls include, butare not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tertiary butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and the like, each of which can beoptionally substituted.

Unless otherwise indicated, the term “optionally substituted,” refers toa group in which none, one, or more than one of the hydrogen atoms hasbeen replaced with one or more group(s) individually and independentlyselected from: cycloalkyl, aryl, heteroaryl, non-aromatic heterocycle,hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo,carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido,C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro,silyl, trihalomethanesulfonyl, and amino, including mono and disubstituted amino groups, and the protected derivatives of amino groups.Such protective derivatives (and protecting groups that can form suchprotective derivatives) are known to those of skill in the art and canbe found in references such as Greene and Wuts, above. In embodiments inwhich two or more hydrogen atoms have been substituted, the substituentgroups can together form a ring.

As used herein, the term “pharmaceutical agent” refers to a chemicalcompound or composition capable of inducing a desired therapeutic effectin a patient. In certain embodiments, a pharmaceutical agent contains anactive agent, which is the agent that induces the desired therapeuticeffect. In certain embodiments, a pharmaceutical agent is a prodrug. Incertain embodiments, a pharmaceutical agent contains inactiveingredients such as carriers, excipients, and the like.

As used herein, the term “therapeutically effective amount” refers to anamount of a pharmaceutical agent sufficient to achieve a desiredtherapeutic effect.

As used herein, the term “pharmaceutically acceptable” refers to aformulation of a compound that does not significantly abrogate thebiological activity, a pharmacological activity and/or other propertiesof the compound when the formulated compound is administered to apatient. In certain embodiments, a pharmaceutically acceptableformulation does not cause significant irritation to a patient.

As used herein, pharmaceutically acceptable derivatives of a compoundinclude salts, esters, enol ethers, enol esters, acetals, ketals,orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydratesor prodrugs thereof. Such derivatives can be readily prepared by thoseof skill in this art using known methods for such derivatization. Thecompounds produced can be administered to animals or humans withoutsubstantial toxic effects and either are pharmaceutically active or areprodrugs. The term “pharmaceutically acceptable salts” refers to thepharmaceutically acceptable and common salts, for example, a baseaddition salt to carboxyl group when the compound has a carboxyl group,or an acid addition salt to amino or basic heterocyclyl when thecompound has an amino or basic heterocyclyl group, including quaternaryammonium salts, prepared from pharmaceutically acceptable non-toxicbases or acids including inorganic or organic bases and inorganic ororganic acids. Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc, and the like. Particularlypreferred are the ammonium, calcium, magnesium, potassium, and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines, and basic ion exchange resins, such as arginine, betaine,caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine, and the like. The term “pharmaceuticallyacceptable salt” further includes all acceptable salts such as acetate,lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate,maleate, bisulfate, mandelate, bitartrate, mesylate, borate,methylbromide, bromide, methylnitrate, calcium edetate, methylsulfate,camsylate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate,N-methylglucamine, citrate, ammonium salt, dihydrochloride, oleate,edetate, oxalate, edisylate, pamoate (embonate), estolate, palmitate,esylate, pantothenate, fumarate, phosphate/diphosphate, gluceptate,polygalacturonate, gluconate, salicylate, glutamate, stearate,glycollylarsanilate, sulfate, hexylresorcinate, subacetate, hydrabamine,succinate, hydrobromide, tannate, hydrochloride, tartrate,hydroxynaphthoate, teoclate, iodide, tosylate, trifluoro acetate,isothionate, triethiodide, lactate, panoate, valerate, and the likewhich can be used as a dosage form for modifying the solubility orhydrolysis characteristics or can be used in sustained release orprodrug formulations. The pharmaceutically acceptable salts of thecomposition of the instant invention include the composition wherein oneof the individual components of the composition is in the form of apharmaceutically acceptable salt, or the composition wherein all of theindividual components are in the form of pharmaceutically acceptablesalts (wherein the salts for each of the components can be the same ordifferent), or a pharmaceutically acceptable salt of the combinedcomponents (i.e., a salt of the composition).

The “pharmaceutically acceptable esters” in the present invention referto non-toxic esters, for example, the pharmaceutically acceptable,common esters on carboxyl group when the compound has a carboxyl group,for example, esters with lower alkyls (for example methyl, ethyl,propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl,neopentyl, cyclopropyl, cyclobutyl, cyclopentyl), aralkyls (for examplebenzyl, phenethyl), lower alkenyls (for example allyl, 2-butenyl), loweralkoxy (lower) alkyls (for example methoxymethyl, 2-methoxyethyl,2-ethoxyethyl), lower alkanoyloxy (lower) alkyls (for exampleacetoxymethyl, pivaloyloxy-methyl, 1-pivaloyloxyethyl), loweralkoxycarbonyl (lower) alkyls (for example methoxycarbonylmethyl,isopropoxycarbonylmethyl), carboxy-lower)alkyls (for examplecarboxymethyl), lower alkoxycarbonyloxy-(lower)alkyls (for example1-(ethoxycarbonyloxy)ethyl, 1-(cyclohexyl-oxycarbonyloxy)ethyl),carbamoyloxy(lower)alkyls (for example carbamoyloxymethyl), phthalidylgroup, (5-substituted-2-oxo-1,3-dioxol-4-yl)methyl (for example(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl), and the like.

The nanosized particles employed in the composition has a medianparticle size of about 1 nm to about 2700 nm, preferably about 200 nm toabout 2200 nm and more preferably about 300 nm to about 2200 nm, andeven more preferably about 300 to about 2100 nm, most preferably about300 nm to about 1000 nm.

As used herein, micronized refers to objects having an average sizeranging from 1 μm to 1000 μm, as measured by light-scattering methods,microscopy, or other appropriate methods. The micronized compositionspreferably have particle size in the range of about 1 μm to 1000 μm,more preferably about 1 μm to 100 μm, even more preferably about 1 μm to50 μm, even more preferably about 1 μm to 25 μm, most preferably about 1μm to 10 μm.

The compounds in the compositions of the present invention includestereoisomers, such as optical isomers, diastereomers and geometricalisomers, or tautomers depending on the mode of substitution. Thecompounds may contain one or more chiral centers and occur as racemates,racemic mixtures and as individual diastereomers, enantiomeric mixturesor single enantiomers, or tautomers, with all isomeric forms beingincluded in the present invention. The present invention is meant tocomprehend all such isomeric forms of the compounds in the compositionsof the present invention, and their mixtures. Therefore, where acompound is chiral, the separate enantiomers, substantially free of theother, are included within the scope of the invention; further includedare all mixtures of the two enantiomers. Also included within the scopeof the invention are polymorphs, hydrates and solvates of the compoundsof the instant invention.

As used herein, a “prodrug” refers to a pharmaceutical agent that isconverted from a less active form into a corresponding more active formin vivo. A prodrug is a compound that, upon in vivo administration, ismetabolized by one or more steps or processes or otherwise converted tothe biologically, pharmaceutically or therapeutically active form of thecompound. To produce a prodrug, the pharmaceutically active compound ismodified such that the active compound will be regenerated by metabolicprocesses. The prodrug can be designed to alter the metabolic stabilityor the transport characteristics of a drug, to mask side effects ortoxicity, to improve the flavor of a drug or to alter othercharacteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, those of skill inthis art, once a pharmaceutically active compound is known, can designprodrugs of the compound (see, e.g., Nogrady (1985) Medicinal ChemistryA Biochemical Approach, Oxford University Press, New York).

As used herein, the term “co-administer” refers to administering morethan one pharmaceutical agent to a patient. In certain embodiments,co-administered pharmaceutical agents are administered together in asingle dosage unit. In certain embodiments, co-administeredpharmaceutical agents are administered separately. In certainembodiments, co-administered pharmaceutical agents are administered atthe same time. In certain embodiments, co-administered pharmaceuticalagents are administered at different times.

As used herein “subject” is an animal, typically a mammal, includinghuman.

As used herein, the term “patient” includes human and animal subjects.

Certain compounds that bind to glucocorticoid receptor and/or modulatean activity of such receptors play a role in health (e.g., normalgrowth, development, and/or absence of disease). In certain embodiments,selective glucocorticoid receptor modulators and/or binding compoundsare useful for treating any of a variety of diseases or conditions.Certain compounds have been previously described as receptor modulators.See e.g., U.S. Pat. Nos. 5,693,646; 6,380,207; 6,506,766; 5,688,810;5,696,133; U.S. Patent Publication No. 20070281959; Zhi, et. al.Bioorganic & Medicinal Chemistry Letters 2000, 10, 415-418; Pooley, et.al., J. Med. Chem. 1998, 41, 3461, the entire disclosures of which areincorporated herein in their entirety.

Estimation of Human Transdermal Dose

The following approach to estimate human dose.

1. Comparison of Toxicity of Anticancer Agents in Different Species

Freireich et al. analyzed toxicity data for 18 cancer chemotherapeuticdrugs in six species, including mouse, rat, hamster, dog, monkey, andhuman. (Freireich et. al., Cancer Chemotherapy Rep. 50:219-244, 1966).On a mg/m² basis the toxic dose was approximately the same in eachspecies. On a mg/kg basis the mouse dose was about 1/12 of the human.This relationship enables one to predict the effective human dose of adrug based on animal data, although there are exceptions and some drugscould show greater potency in one species versus another. Using theabove relationship, the about 200 mg/kg oral dose of fluasterone inmouse translates into about 1200 mg daily in the human and about 5 mg/kgtransdermal dose translates into about 28 mg human dose. Thecompositions herein can be formulated in the transdermal dosage range ofabout 0.01 mg/kg to about 100 mg/kg; preferably about 0.01 mg/kg toabout 75 mg/kg; more preferably about 1 mg/kg to about 50 mg/kg.However, the daily dose can vary based on the animal and the medicalcondition being treated. For humans, the transdermal dosage can be inthe ranges of about 0.01 to about 50 mg/kg, preferably, about 0.01 toabout 25 mg/kg and more preferably, about 0.01 to about 10 mg/kg andeven more preferably about 0.01 to about 5 mg/kg.2. Estimation of Human Dose Based on Plasma Levels of FluasteroneFollowing Oral Administration to Humans or MiceAn oral single dose PK experiment in mice following administration of200 mg/kg fluasterone was performed. The calculated AUCO-24 was 2,242(ngXh/mL). A PK study was also performed in 12 human volunteers as partof a phase I fluasterone study. The volunteers received either 800 mg or1600 mg fluasterone orally as a daily dose. The AUCO-24/mg dose wascalculated. The calculated AUCO-24 for the 1200 mg dose was 2,316(ngXh/mL), which suggests that the 1200 mg human dose, as predicted byFreireich et al. (Supra), is bioequivalent to 200 mg/kg in the mouse.3. Efficacy of Oral about 1200 mg Fluasterone Dose inHypertriglyceridemic PatientsThe lowest effective oral dose of fluasterone which lowers fastingplasma triglycerides in diabetic mice is about 200 mg/kg, whichtranslates into an oral human dose of about 1200 mg. In a phase I/IItrial in hypertriglyceridemic patients we found that a dose of about1200 mg significantly lowered fasting plasma triglyceride levels.The data indicate that, as a reasonable guide, the approach of Freireichet al. can be used to predict human dose of fluasterone. Based on theefficacy of about 5 mg/kg administered transdermally to mice, a probablehuman dose range for transdermal delivery would be about 25 mg to about50 mg.

EXAMPLES Example 1 Solubility of Compounds of Formula I

Fluasterone: The solubility of fluasterone in various solvents wasdetermined by adding a specific volume of solvent to a weighed amount offluasterone in a screw-capped glass vial and shaking vigorously forseveral minutes. Incremental volumes of solvent were added, followed byvigorous shaking, until the fluasterone completely dissolved.

Alternatively solubility was determined by adding a specific volume ofsolvent to a weighed amount of fluasterone in a screw-capped glass vialand warming the vial on a hot plate with vigorous shaking until thefluasterone dissolved. Fluasterone was considered soluble if it remainedin solution after at least a month at room temperature. Although theabove procedures may not give precise solubilities, they should give aclose approximation of maximum fluasterone solubility.

TABLE 2 Fluasterone solubility Solvent Solubility Ethanol 6.6 mg/mLPolyethylene glycol 400 (PEG-400) 1.1 mg/mL Propylene glycol >0.5 mg/mLLimonene 22.6 mg/mL Dimethyl sulfoxide (DMSO) 3.6 mg/mL IsopropylMyristate 16 mg/mL

Example 2 Solubility in Mixed Solvents

In these experiments fluasterone solubility was determined by addingfluasterone to a measured volume of solvent in a screw-capped glassvial. The vial was warmed on a hot plate with vigorous shaking until thefluasterone dissolved. Fluasterone was considered soluble if it remainedin solution after at least a month at room temperature.

Solubility in 10% Tween 80-90% Ethanol (v/v): 21.4 mg of micronizedfluasterone was weighed in a screw-capped glass vial and about 2.14 mLof a mixture of 10% Tween 80 (Sigma ultra) and 90% ethanol (ACS/USPgrade) was added. The vial was warmed on a hot plate until thefluasterone dissolved. A few fine crystals of fluasterone were observedafter standing 3 weeks at room temperature. Solubility is slightly lessthan 10 mg/mL.Solubility in 15% Tween 80-85% Ethanol (v/v): 20.0 mg of micronizedfluasterone was weighed in a screw-capped glass vial and 2.0 mL of amixture of 15% Tween 80 and 85% ethanol added, and the vial was warmedon a hot plate until the fluasterone dissolved. A few fine crystals offluasterone were observed after standing 3 weeks at room temperature.Solubility is slightly less than 10 mg/mL.Solubility in 20% Tween 80-80% Ethanol (v/v): 18.5 mg of micronizedfluasterone was weighed in a screw-capped glass vial and about 1.85 mLof a mixture of 20% Tween 80 and 80% ethanol was added. Fluasterone wasdissolved by warming as described. The solution remained visibly clearafter 2 months at room temperature. 28.5 mg of micronized fluasteronewas weighed in a screw-capped glass vial and about 2.38 mL of a mixtureof 20% Tween 80 and 80% ethanol was added (12 mg/mL). Fluasterone wasdissolved by warming as described. After 3 days at room temperature somefluasterone crystals were seen. Solubility of fluasterone in 20% Tween80 and 80% ethanol is greater than 10 mg/mL and less than 12 mg/mL.Solubility in 50% Tween 80-50% Ethanol (v/v): 28.3 mg of micronizedfluasterone was weighed in a screw-capped glass vial and 1.8 mL of amixture of 50% Tween 80 and 50% ethanol was added (16 mg/mL).Fluasterone was dissolved by warming. A few crystals of fluasterone wereobserved after 1 month at room temperature. 28.8 mg of micronizedfluasterone was weighed in a screw-capped glass vial and about 2.05 mLof a mixture of 50% Tween 80 and 50% ethanol was added (14 mg/mL).Fluasterone was dissolved by warming. After 7 weeks at room temperaturethe solution is visibly clear. Solubility is greater than 14 mg/mL andless than 16 mg/mL.Solubility of Fluasterone in Cetyl Alcohol Ethanol (10%:90% w/w)Mixture: 29.9 mg of micronized fluasterone was weighed in a screw-cappedglass vial and 3 mL of a mixture of 10% cetyl alcohol:90% ethanol added.The vial was warmed on a hot plate until the fluasterone dissolved.After 2 days at room temperature, some fluasterone crystals were seen.Solubility of fluasterone is somewhat less than 10 mg/mL.Solubility of Fluasterone in Cetyl Alcohol:Ethanol (20%:80% w/w)Mixture: 32.3 mg of micronized fluasterone was weighed in a screw-cappedglass vial and 2.7 mL of 20% cetyl alcohol:80% ethanol was added (12mg/mL). The vial was warmed on a hot plate until the fluasteronedissolved. After about 5 weeks at room temperature, a few small crystalsof fluasterone were observed. Solubility of fluasterone is slightly lessthan 12 mg/mL. A solution of fluasterone (10 mg/mL) was also prepared ina mixture of 22% cetyl alcohol:78% ethanol. After about 1 month at roomtemperature the solution was completely clear.

Solubility of fluasterone is greater than 10 mg/mL and less than 12mg/mL, which is similar to Tween 80:ethanol mixtures.

Example 3 Dose-Response with Orally Administered Fluasterone on12-O-tetradecanoylphorbol-13-acetate (TPA)-Induced DNA Synthesis inMouse Epidermis

In order to determine the lower range of effective oral dose ofFluasterone which abolishes 12-O-tetradecanoylphorbol-13-acetate(TPA)-stimulated epidermal hyperplasia in mouse skin the followingexperiment was performed.

Fluasterone suspensions were prepared using sesame oil as vehicle.Micronized fluasterone was obtained from Pharmaceutics International,Inc. Five suspensions were prepared—100 mg/kg (13 mg/mL), 200 mg/kg(26.5 mg/mL), 400 mg/kg (52.4 mg/mL), 800 mg/kg (105.5 mg/mL), and 1200mg/kg (158.3 mg/mL). The fluasterone for each suspension was weighed,placed into a plastic vial, and the required volume of sesame oil wasadded to the vial. The fluasterone was suspended in the sesame oil withthe use of a Tekmar Tissumizer. Magnetic stirrer bars were added to thevarious suspensions which were stirred on a magnetic stirrer prior totreatment. Control and TPA groups received sesame oil alone.

Female CD-1 mice were obtained from Charles River Laboratories,Wilmington, Mass. at 35-37 days of age.

The mice were housed three per cage in plastic shoebox cages on Alphacelbedding with 12 hours of alternating light and dark in the CAF AnimalFacility. The mice had ad libitum access to Purina 5015 chow andacidified tap water (pH≦2.6). The mice were shaved, weighed anddistributed into groups of three.

Control

The mice were treated orally with 0.2 mL of sesame oil. One hour aftertreatment with sesame oil, the mice were treated topically with 0.2 mLof acetone.

TPA

The mice were treated orally with 0.2 mL of sesame oil. One hour aftertreatment with sesame oil, the mice were treated topically with 2 μg ofTPA in 0.2 mL of acetone.

-   Fluasterone 100 mg/kg The mice were treated with 0.2 mL of a    suspension of micronized fluasterone to give a dose of 100 mg/kg.    One hour after treatment with fluasterone, the mice were treated    topically with 2 μg of TPA in 0.2 mL of acetone.-   Fluasterone 200 mg/kg The mice were treated with 0.2 mL of a    suspension of micronized fluasterone to give a dose of 200 mg/kg.    One hour after treatment with fluasterone, the mice were treated    topically with 2 μg of TPA in 0.2 mL of acetone.-   Fluasterone 400 mg/kg The mice were treated with 0.2 mL of a    suspension of micronized fluasterone to give a dose of 400 mg/kg.    One hour after treatment with fluasterone, the mice were treated    topically with 2 μg of TPA in 0.2 mL of acetone.-   Fluasterone 800 mg/kg The mice were treated with 0.2 mL of a    suspension of micronized fluasterone to give a dose of 800 mg/kg.    One hour after treatment with fluasterone, the mice were treated    topically with 2 μg of TPA in 0.2 mL of acetone.-   Fluasterone 1200 mg/kg The mice were treated with 0.2 mL of a    suspension of micronized fluasterone to give a dose of 1200 mg/kg.    One hour after treatment with fluasterone, the mice were treated    topically with 2 μg of TPA in 0.2 mL of acetone.

The mice were sacrificed 20 hours after treatment by an overdose of CO₂.Twenty minutes prior to sacrifice, the mice were injected with 60 μCi of[³H]thymidine (Amersham). The mice were treated with a depilatory toremove any residual hair. A 2×2 cm² piece of skin was excised, placed inice water for 30 seconds, then in 55° C. water for 30 seconds, then inice water again for 30 seconds. The epidermis was s raped off using ascalpel and the scrapings were placed into ice cold 0.4N TCA. Thescrapings were homogenized using a Tekmar Tissumizer (80% power for 30seconds). The homogenates were centrifuged for 20 minutes at 3,000×g.The precipitates were washed 3× with 0.2N TCA. The TCA was removed andthe precipitates were stored at −20° C. overnight. The next day, theprecipitates were washed 2× with absolute ethanol. The DNA in eachsample was hydrolyzed with 0.5N TCA for 30 minutes at 90°. The tubeswere centrifuged for 20 minutes at 3000×g. A 0.2 mL aliquot of eachhydrolysate was counted in a LKB Rackbeta scintillation counter usingScintiverse II BD as the counting medium. DNA content was determined bythe Burton diphenylamine assay.

Fluasterone Suspensions:

-   -   100 mg/kg: 26.0 mg was suspended in 2 mL of sesame oil) (13        mg/mL)    -   200 mg/kg: 61.0 mg was suspended in 2.3 mL of sesame oil (26.5        mg/mL)    -   400 mg/kg: 125.8 mg was suspended in 2.4 mL of sesame oil (52.4        mg/mL)    -   800 mg/kg: 211 mg was suspended in 2.0 mL of sesame oil (105.5        mg/mL)    -   1200 mg/kg: 316.6 mg was suspended in 2.0 mL of sesame oil        (158.3 mg/mL)

TABLE 3 Average body weights of mice 800 1200 Control TPA 100 mg/kg 200mg/kg 400 mg/kg mg/kg mg/kg 25.5 ± 26.1 ± 26.5 ± 0.3 26.3 ± 0.4 26.2 ±0.3 26.4 ± 26.4 ± 0.9 1.0 0.3 0.4

TABLE 4 Results of TPA induced DNA synthesis μg DNA cpm/μg DNA GroupControl 4.7 ± 1.0 156.1 ± 33.6  TPA 14.1 ± 1.3  641.5 ± 115.2Fluasterone Groups:  100 mg/kg 15.5 ± 3.0  539.8 ± 75.9   200 mg/kg 6.0± 1.9 185.0 ± 38.2   400 mg/kg 6.6 ± 2.6 208.8 ± 144.2  800 mg/kg 6.2 ±1.0 634.3 ± 151.6 1200 mg/kg 6.3 ± 1.5 850.0 ± 256.0

This experiment demonstrates that 200 mg/kg p.o. is the lowest testeddose which inhibits TPA-stimulated epidermal [³H] thymidineincorporation (cpm/μg DNA) as well as the epidermal DNA content of a 2×2cm² section of mouse skin (μg DNA). As the dose of fluasterone isincreased, the cpm/μg DNA increases while the μg DNA value remainsdepressed. 200 mg/kg p.o. fluasterone is also the lowest effective dosewhich significantly lowers fasting plasma glucose and triglyceridelevels in diabetic mice. As discussed in page 7, this is presumably froma decrease in the endogenous thymidine pool size as a consequence ofG6PDH inhibition

Example 4 Dose Response Efficacy of Transdermally AdministeredFluasterone in Inhibiting TPA-Stimulated Epidermal DNA Synthesis inMouse Epidermis

The solubility of fluasterone in various formulations have been testedfor efficacy.

The following fluasterone solutions, and one suspension, have beentested in three TPA experiments.

1. A solution of fluasterone in carbitol at 12.7 mg/mL.

2. A solution of fluasterone in Cremophor EL (polyethoxylated castoroil) at 12.4 mg/mL.

3. A solution of fluasterone in a 50:50 mixture (v/v) of ethanol andTween 80 at 10 mg/mL.

4. A solution of fluasterone in a 50:50 mixture (v/v) of ethanol andCremophor EL at 10 mg/mL.

5. A solution of fluasterone in an 81:15:4 mixture of ethanol:R-(+)-limonene:isopropyl myristate (v/v/v) at 10 mg/mL.

6. A suspension of micronized fluasterone in a mixture of 76:19:5ethanol:water:Tween-80 (v/v/v) at 10 mg/mL.

These solutions and one suspension have all suppressed TPA-stimulatedepidermal [³H] thymidine and epidermal DNA content at a dose of 2.5mg/kg, which is the same potency of fluasterone following s.c.administration.

The Effect of Topically-Administered Fluasterone Dissolved in EitherCarbitol or Cremophor EL on TPA-Induced DNA Synthesis in MouseEpidermis:

This experiment demonstrates that transdermally applied fluasterone insolution in either carbitol or Cremophor EL, at a dose of 2.5 mg/kg,inhibits TPA-stimulated epidermal [³H] thymidine incorporation (cpm/μg)as well as the epidermal DNA content of a 2×2 cm² section of mouse skin(μg DNA). This is the same potency seen with s.c.-injected fluasteronein this assay.

Treatment with the carbitol formulation produced a greater overshoot in[³H] thymidine incorporation at 5 mg/kg (501±47.2 vs. 132±21.7),suggesting that fluasterone may be more bioavailable when dissolved incarbitol vs. Cremophor EL as the dose of fluasterone is increased.

Methods: Female CD-1 mice, 40-43 days old, were obtained from CharlesRiver Laboratories, Kingston, N.Y. The mice were housed two per cage inplastic shoebox cages on Absorbdri bedding with 12 hours of alternatinglight and dark in the Central Animal Facility. The mice had ad libitumaccess to Purina 5015 chow and acidified tap water (pH≦2.6). The micewere allowed to acclimate to the facility for approximately two weeksprior to use in an experiment.

Micronized fluasterone was used. 24.1 mg of micronized fluasterone wasweighed using a Mettler AE-50 balance, transferred to a glassscintillation vial, and dissolved in 1.9 mL of carbitol for the 5 mg/kg,2.5 mg/kg, 1.9 mg/kg and 1.25 mg/kg fluasterone/carbitol groups. 23.6 mgof micronized fluasterone was weighed and dissolved in 1.9 mL ofCremophor EL for the 5 mg/kg, 2.5 mg/kg, 1.9 mg/kg and 1.25 mg/kgfluasterone/Cremophor EL groups.

The mice were weighed, marked with magic marker on the tail, and shavedon the back and the abdomen one day prior to treatment. Only those miceshowing no hair regrowth were used in the experiment. Fluasterone wasapplied to the shaved abdomen one hour before TPA was applied to theshaved back. The mice were anesthetized and on their backs prior totopical application of TPA.

The mice were treated as follows, with two mice in each experimentalgroup.

Carbitol Groups

Control: The mice were anesthetized with Isoflurane, and then were givenan intramuscular injection of 0.1 ml solution of ketamine (50 mg/kg),xylazine (10 mg/kg) and atropine (0.1 mg/kg). Approximately 10 minutesafter the i.m. Injection (after the mice could not turn over when placedon their backs), the mice were treated with 10 μL of carbitol. The micewere anesthetized and on their backs for approximately 30 minutes afterapplication of carbitol. One hour after the application of carbitol, 0.2ml of acetone was applied to the shaved area on the back of each mouse.

TPA: The mice were anesthetized with Isoflurane, and then were given anintramuscular injection of 0.1 mL solution of ketamine (50 mg/kg),xylazine (10 mg/kg) and atropine (0.1 mg/kg). Approximately 10 minutesafter the i.m. injection (after the mice could not turn over when placedon their backs), the mice were treated with 10 μL of carbitol. The micewere anesthetized and on their backs for approximately 30 minutes afterapplication of carbitol. One hour after the application of carbitol, 2μg of TPA dissolved in 0.2 mL of acetone was applied to the shaved areaon the back of each mouse.5 mg/kg Fluasterone: The mice were anesthetized with Isoflurane, andthen were given an intramuscular injection of 0.1 mL solution ofketamine (50 mg/kg), xylazine (10 mg/kg) and atropine (0.1 mg/kg).Approximately 10 minutes after the i.m. injection (after the mice couldnot turn over when placed on their backs), the mice were treated with 10μL of a 12.5 mg/mL fluasterone/carbitol solution to give a dose of 5mg/kg. The mice were anesthetized and on their backs for approximately30 minutes after application of fluasterone/carbitol solution. One hourafter the application of fluasterone/carbitol solution, 2 μg of TPAdissolved in 0.2 mL of acetone was applied to the shaved area on theback of each mouse.2.5 mg/kg Fluasterone The mice were anesthetized with Isoflurane, andthen were given an intramuscular injection of 0.1 mL solution ofketamine (50 mg/kg), xylazine (10 mg/kg) and atropine (0.1 mg/kg).Approximately 10 minutes after the i.m. injection (after the mice couldnot turn over when placed on their backs), the mice were treated with 5μL of a 12.5 mg/mL fluasterone/carbitol solution to give a dose of 2.5mg/kg. The mice were anesthetized and on their backs for approximately30 minutes after application of fluasterone/carbitol solution. One hourafter the application of fluasterone/carbitol solution, 2 μg of TPAdissolved in 0.2 mL of acetone was applied to the shaved area on theback of each mouse.1.9 mg/kg Fluasterone The mice were anesthetized with Isoflurane, andthen were given an intramuscular injection of 0.1 mL solution ofketamine (50 mg/kg), xylazine (10 mg/kg) and atropine (0.1 mg/kg).Approximately 10 minutes after the i.m. injection (after the mice couldnot turn over when placed on their backs), the mice were treated with3.8 μL of a 12.5 mg/mL fluasterone/carbitol solution to give a dose of1.9 mg/kg. The mice were anesthetized and on their backs forapproximately 30 minutes after application of fluasterone/carbitolsolution. One hour after the application of fluasterone/carbitolsolution, 2 μg of TPA dissolved in 0.2 mL of acetone was applied to theshaved area on the back of each mouse.1.25 mg/kg Fluasterone The mice were anesthetized with Isoflurane, andthen were given an intramuscular injection of 0.1 mL solution ofketamine (50 mg/kg), xylazine (10 mg/kg) and atropine (0.1 mg/kg).Approximately 10 minutes after the i.m. injection (after the mice couldnot turn over when placed on their backs), the mice were treated with2.5 μL of a 12.5 mg/mL fluasterone/carbitol solution to give a dose of1.25 mg/kg. The mice were anesthetized and on their backs forapproximately 30 minutes after application of fluasterone/carbitolsolution. One hour after the application of fluasterone/carbitolsolution, 2 μg of TPA dissolved in 0.2 mL of acetone was applied to theshaved area on the back of each mouse.Cremophor EL Groups:Control: The mice were anesthetized with Isoflurane, and then were givenan intramuscular injection of 0.1 mL solution of ketamine (50 mg/kg),xylazine (10 mg/kg) and atropine (0.1 mg/kg). Approximately 10 minutesafter the i.m. injection (after the mice could not turn over when placedon their backs), the mice were treated with 10 μL of Cremophor EL. Themice were anesthetized and on their backs for approximately 30 minutesafter application of Cremophor EL. One hour after the application ofCremophor EL, 0.2 mL of acetone was applied to the shaved area on theback of each mouse.TPA: The mice were anesthetized with Isoflurane, and then were given anintramuscular injection of 0.1 mL solution of ketamine (50 mg/kg),xylazine (10 mg/kg) and atropine (0.1 mg/kg). Approximately 10 minutesafter the i.m. injection (after the mice could not turn over when placedon their backs), the mice were treated with 10 μL of Cremophor EL. Themice were anesthetized and on their backs for approximately 30 minutesafter application of Cremophor EL. One hour after the application ofCremophor EL, 2 μg of TPA dissolved in 0.2 mL of acetone was applied tothe shaved area on the back of each mouse.5 mg/kg Fluasterone: The mice were anesthetized with Isoflurane, andthen were given an intramuscular injection of 0.1 mL solution ofketamine (50 mg/kg), xylazine (10 mg/kg) and atropine (0.1 mg/kg).Approximately 10 minutes after the i.m. injection (after the mice couldnot turn over when placed on their backs), the mice were treated with 10μL of a 12.5 mg/mL fluasterone/Cremophor EL solution to give a dose of 5mg/kg. The mice were anesthetized and on their backs for approximately30 minutes after application of fluasterone/Cremophor EL solution. Onehour after the application of fluasterone/Cremophor EL solution, 2 μg ofTPA dissolved in 0.2 mL of acetone was applied to the shaved area on theback of each mouse.2.5 mg/kg Fluasterone: The mice were anesthetized with Isoflurane, andthen were given an intramuscular injection of 0.1 mL solution ofketamine (50 mg/kg), xylazine (10 mg/kg) and atropine (0.1 mg/kg).Approximately 10 minutes after the i.m. injection (after the mice couldnot turn over when placed on their backs), the mice were treated with 5μL of a 12.5 mg/mL fluasterone/Cremophor EL solution to give a dose of2.5 mg/kg. The mice were anesthetized and on their backs forapproximately 30 minutes after application of fluasterone/Cremophor ELsolution. One hour after the application of fluasterone/Cremophor ELsolution, 2 μg of TPA dissolved in 0.2 mL of acetone was applied to theshaved area on the back of each mouse.1.9 mg/kg Fluasterone: The mice were anesthetized with Isoflurane, andthen were given an injection of 0.1 mL solution of ketamine (50 mg/kg),xylazine (10 mg/kg) and atropine (0.1 mg/kg). Approximately 10 minutesafter the i.m. injection (after the mice could not turn over when placedon their backs), the mice were treated with 3.8 μL of a 12.5 mg/mLfluasterone/Cremophor EL solution to give a dose of 1.9 mg/kg. The micewere anesthetized and on their backs for approximately 30 minutes afterapplication of fluasterone/Cremophor EL solution. One hour after theapplication of fluasterone/Cremophor EL solution, 2 μg of TPA dissolvedin 0.2 mL of acetone was applied to the shaved area on the back of eachmouse.1.25 mg/kg Fluasteorne: The mice were anesthetized with Isoflurane, andthen were given an intramuscular injection of 0.1 mL solution ofketamine (50 mg/kg), xylazine (10 mg/kg) and atropine (0.1 mg/kg).Approximately 10 minutes after the i.m. injection (after the mice couldnot turn over when placed on their backs), the mice were treated with2.5 μL of a 12.5 mg/mL fluasterone/Cremophor EL solution to give a doseof 1.25 mg/kg. The mice were anesthetized and on their backs forapproximately 30 minutes after application of fluasterone/Cremophor ELsolution. One hour after the application of fluasterone/Cremophor ELsolution, 2 μg of TPA dissolved in 0.2 mL of acetone was applied to theshaved area on the back of each mouse.

The mice were sacrificed 20 hours after treatment by an overdose of CO₂.Twenty minutes prior to sacrifice, the mice were injected with 60 μCi of[³H] thymidine). The mice were treated with a depilatory to remove anyresidual hair. A piece of skin (approximately 2×2 cm²) was excised,placed in ice water for 30 seconds, then in 55° water for 30 seconds,then in ice water again for 30 seconds. The epidermis was scraped offusing a scalpel and the scrapings were placed into ice cold 0.4N TCA.The scrapings were homogenized using a Tekmar Tissumizer (80% power for30 seconds). The homogenates were centrifuged for 20 minutes at 3000×g.The precipitates were washed 4× with 0.2N TCA, and 2× with absoluteethanol. The DNA was hydrolyzed with 0.5N TCA for 30 minutes at 90°. Thetubes were centrifuged for 20 minutes at 3000×g. 0.2 mL aliquots of thesupernatants were counted in an LKB 1209 Rackbeta scintillation counter,using Scintiverse BD as counting medium. DNA content was determined bythe Burton diphenylamine assay.

TABLE 5 Body weight of mice in the carbitol group Control TPA 5 mg/kg2.5 mg/kg 1.9 mg/kg 1.25 mg/kg 26.0 ± 0.5 24.8 ± 0.2 25.8 ± 0.4 25.5 ±0.2 25.3 ± 0.2 25.3 ± 0.4

TABLE 6 Body weight of mice in the Cremophor EL group Control TPA 5mg/kg 2.5 mg/kg 1.9 mg/kg 1.25 mg/kg 26.0 ± 0.4 25.4 ± 0.6 25.3 ± 0.125.6 ± 0.1 25.6 ± 0.1 25.4 ± 0.0

TABLE 7 Effect of fluasterone on TPA-induced DNA synthesis correctedGroup cpm μg DNA/0.2 mL cpm cpm/μg DNA Carbitol Groups Control 606 8.6571 66.4 461 9.1 426 46.8  56.6 ± 13.9 TPA 6,764 20.3 6,729 331.5 7,53121.2 7,496 353.6 342.6 ± 15.6   5 mg/kg 5,373 10.0 5,338 533.8 5,08010.8 5,045 467.1 500.5 ± 47.2  2.5 mg/kg 651 9.5 616 64.8 684 8.8 64973.8 69.3 ± 6.3  1.9 mg/kg 6,767 18.4 6,732 365.9 2,722 11.8 2,687 227.7296.8 ± 97.7 1.25 mg/kg 6,086 22.4 6,051 270.1 10,205 29.8 10,170 341.3287.9 ± 25.2 Cremophor EL Groups Control 903 8.7 868 99.8 782 8.5 74787.9 93.9 ± 8.4 TPA 16,877 28.7 16,842 586.8 13,884 30.4 13,849 455.6521.2 ± 92.8   5 mg/kg 1,015 8.4 980 116.7 1,686 11.2 1,651 147.4 132.1± 21.7  2.5 mg/kg 590 8.6 555 64.5 1,359 10.1 1,324 131.1  97.8 ± 47.1 1.9 mg/kg 2,514 15.4 2,479 161.0 5,468 20.1 5,433 270.3 215.7 ± 77.31.25 mg/kg 14,290 28.1 14,255 507.3 6,208 14.9 6,173 414.3 460.1 ± 65.8

Example 5 The Effect of Topically Applied-Fluasterone Dissolved inEither Cremophor EL:ethanol (50:50 v/v) or Tween 80:Ethanol (50:50 v/v)on TPA-Induced DNA Synthesis in Mouse Epidermis

In the Tween 80:ethanol group, a dose of 2.5 mg/kg suppressed theTPA-stimulated increase in epidermal DNA content (μg DNA). With respectto [³H] thymidine incorporation (cpm/μg DNA), the 2.5 mg/kg dosesuppressed this value in 1 of 3 mice, whereas the other 2 mice showedthe artifactual increase. The lowest effective dose is about 2.5 mg/kgor somewhat lower.

In the Cremophor EL:ethanol group, treatment with this formulationgreatly enhanced the effect of TPA in stimulating [³H] thymidineincorporation and epidermal DNA content when compared to the Tween80:ethanol group. Cremophor EL had been used to formulate the drugpaclitaxel for i.v. administration and is believed to cause acutehypersensitivity reactions. Cremophor EL has been replaced by Tween-80to formulate paclitaxel. As such, Cremophor EL may not be the bestsolvent for formulating fluasterone.

Methods: Female CD-1 mice, 40-43 days old, were obtained from CharlesRiver Laboratories, Kingston, N.Y. The mice were housed two per cage inplastic shoebox cages on Absorbdri bedding with 12 hours of alternatinglight and dark. The mice had ad libitum access to Purina 5015 chow andacidified tap water (pH≦2.6). The mice were allowed to acclimate to thefacility for approximately one week prior to use in an experiment.

Micronized 8354 was used. 34.1 mg of micronized 8354 was weighed using aMettler AE-50 balance, transferred to a glass scintillation vial, anddissolved in 3.4 mL of Cremophor EL:ethanol (50:50 v/v) for the 5 mg/kg,2.5 mg/kg, and 1.9 mg/kg fluasterone:Cremophor EL:ethanol groups. 32.1mg of micronized fluasterone was weighed and dissolved in 3.2 mL ofTween 80:ethanol solution (50:50, v/v) for the 5 mg/kg, 2.5 mg/kg, and1.9 mg/kg fluasterone:Tween 80:ethanol groups.

The mice were weighed, marked with magic marker on the tail, and shavedon the back and the abdomen two days prior to treatment. Only those miceshowing no hair regrowth were used in the experiment.

The mice were treated as follows, with two mice in each experimentalgroup

Cremophor EL:Ethanol Groups:

Control: The mice were anesthetized with Isoflurane, and then were givenan intramuscular injection of 0.1 mL solution of ketamine (50 mg/kg) andxylazine (10 mg/kg). Approximately 10 minutes after the i.m. injection(after the mice could not turn over when placed on their backs), themice were treated with 12.5 μL of Cremophor EL:ethanol vehicle. The micewere anesthetized and on their backs for approximately 30 minutes afterapplication of Cremophor EL:ethanol vehicle. One hour after theapplication of Cremophor EL:ethanol solution, 0.2 mL of acetone wasapplied to the shaved area on the back of each mouse.TPA: The mice were anesthetized with Isoflurane, and then were given anintramuscular injection of 0.1 mL solution of ketamine (50 mg/kg) andxylazine (10 mg/kg). Approximately 10 minutes after the i.m. injection(after the mice could not turn over when placed on their backs), themice were treated with 12.5 μL of Cremophor EL:ethanol solution. Themice were anesthetized and on their backs for approximately 30 minutesafter application of Cremophor EL:ethanol solution. One hour after theapplication of Cremophor EL:ethanol solution, 2 μg of TPA dissolved in0.2 mL of acetone was applied to the shaved area on the back of eachmouse.5 mg/kg Fluasterone The mice were anesthetized with Isoflurane, and thenwere given an intramuscular injection of 0.1 mL solution of ketamine (50mg/kg) and xylazine (10 mg/kg). Approximately 10 minutes after the i.m.injection (after the mice could not turn over when placed on theirbacks), the mice were treated with 12.5 μL of a 10 mg/mLfluasterone:Cremophor EL:ethanol solution to give a dose of 5 mg/kg. Themice were anesthetized and on their backs for approximately 30 minutesafter application of Cremophor EL:ethanol solution. One hour after theapplication of Cremophor EL:ethanol solution, 2 μg of TPA dissolved in0.2 mL of acetone was applied to the shaved area on the back of eachmouse.2.5 mg/kg Fluasterone The mice were anesthetized with Isoflurane, andthen were given an intramuscular injection of 0.1 mL solution ofketamine (50 mg/kg) and xylazine (10 mg/kg). Approximately 10 minutesafter the i.m. injection (after the mice could not turn over when placedon their backs), the mice were treated with 6.25 μL of a 10 mg/mLfluasterone:Cremophor EL:ethanol solution to give a dose of 2.5 mg/kg.The mice were anesthetized and on their backs for approximately 30minutes after application of Cremophor EL:ethanol solution. One hourafter the application of Cremophor EL:ethanol solution, 2 μg of TPAdissolved in 0.2 mL of acetone was applied to the shaved area on theback of each mouse.1.9 mg/kg Fluasterone The mice were anesthetized with Isoflurane, andthen were given an intramuscular injection of 0.1 mL solution ofketamine (50 mg/kg) and xylazine (10 mg/kg). Approximately 10 minutesafter the i.m. injection (after the mice could not turn over when placedon their backs), the mice were treated with 4.8 μL of a 10 mg/mLfluasterone:Cremophor EL:ethanol solution to give a dose of 1.9 mg/kg.The mice were anesthetized and on their backs for approximately 30minutes after application of Cremophor EL:ethanol solution. One hourafter the application of Cremophor EL:ethanol solution, 2 μg of TPAdissolved in 0.2 mL of acetone was applied to the shaved area on theback of each mouse.Tween-80/Ethanol Groups:Control: The mice were anesthetized with Isoflurane, and then were givenan intramuscular injection of 0.1 mL solution of ketamine (50 mg/kg) andxylazine (10 mg/kg). Approximately 10 minutes after the i.m. injection(after the mice could not turn over when placed on their backs), themice were treated with 12.5 μL of Tween 80:ethanol solution. The micewere anesthetized and on their backs for approximately 30 minutes afterapplication of Tween 80:ethanol. One hour after the application of Tween80:ethanol, 0.2 mL of acetone was applied to the shaved area on the backof each mouse.TPA: The mice were anesthetized with Isoflurane, and then were given anintramuscular injection of 0.1 mL solution of ketamine (50 mg/kg) andxylazine (10 mg/kg). Approximately 10 minutes after the i.m. injection(after the mice could not turn over when placed on their backs), themice were treated with 12.5 μL of Tween 80:ethanol solution. The micewere anesthetized and on their backs for approximately 30 minutes afterapplication of Tween 80:ethanol solution. One hour after the applicationof Tween 80:ethanol, 2 μg of TPA dissolved in 0.2 mL of acetone wasapplied to the shaved area on the back of each mouse.5 mg/kg Fluasterone The mice were anesthetized with Isoflurane, and thenwere given an intramuscular injection of 0.1 mL solution of ketamine (50mg/kg) and xylazine (10 mg/kg). Approximately 10 minutes after the i.m.injection (after the mice could not turn over when placed on theirbacks), the mice were treated with 12.5 μL of a 10 mg/mLfluasterone:Tween 80:ethanol solution to give a dose of 5 mg/kg. Themice were anesthetized and on their backs for approximately 30 minutesafter application of Tween 80:ethanol solution. One hour after theapplication of Tween 80:ethanol solution, 2 μg of TPA dissolved in 0.2mL of acetone was applied to the shaved area on the back of each mouse.2.5 mg/kg Fluasterone The mice were anesthetized with Isoflurane, andthen were given an intramuscular injection of 0.1 mL solution ofketamine (50 mg/kg) and xylazine (10 mg/kg). Approximately 10 minutesafter the i.m. injection (after the mice could not turn over when placedon their backs), the mice were treated with 6.25 μL of a 10 mg/mLfluasterone:Tween 80:ethanol solution to give a dose of 2.5 mg/kg. Themice were anesthetized and on their backs for approximately 30 minutesafter application of Tween 80:ethanol solution. One hour after theapplication of Tween 80:ethanol solution, 2 μg of TPA dissolved in 0.2mL of acetone was applied to the shaved area on the back of each mouse.1.9 mg/kg Fluasterone The mice were anesthetized with Isoflurane, andthen were given an intramuscular injection of 0.1 mL solution ofketamine (50 mg/kg) and xylazine (10 mg/kg). Approximately 10 minutesafter the i.m. injection (after the mice could not turn over when placedon their backs), the mice were treated with 4.8 μL of a 10 mg/mLfluasterone:Tween 80:ethanol solution to give a dose of 1.9 mg/kg. Themice were anesthetized and on their backs for approximately 30 minutesafter application of Tween 80:ethanol solution. One hour after theapplication of Tween 80:ethanol solution, 2 μg of TPA dissolved in 0.2mL of acetone was applied to the shaved area on the back of each mouse.

The mice were sacrificed 20 hours after treatment by an overdose of CO₂.Twenty minutes prior to sacrifice, the mice were injected with 60 μCi of[³H] thymidine. The mice were treated with a depilatory to remove anyresidual hair. A piece of skin (approximately 2×2 cm²) was excised,placed in ice water for 30 seconds, then in 55° water for 30 seconds,then in ice water again for 30 seconds. The epidermis was scraped offusing a scalpel and the scrapings were placed into ice cold 0.4N TCA.The scrapings were homogenized using a Tekmar Tissumizer (80% power for30 seconds). The homogenates were centrifuged for 20 minutes at 3000×g.The precipitates were washed 4× with 0.2N TCA, and 2× with absoluteethanol. The DNA was hydrolyzed with 0.5N TCA for 30 minutes at 90°. Thetubes were centrifuged for 20 minutes at 3000×g. 0.2 mL aliquots of thesupernatants were counted in an LKB 1209 Rackbeta scintillation counter,using Scintiverse BD as counting medium. DNA content was determined bythe Burton diphenylamine assay.

TABLE 8 Body weight of mice in the cremophor EL:ethanol solution GroupsControl TPA 5 mg/kg 2.5 mg/kg 1.9 mg/kg 23.5 ± 0.2 24.3 ± 0.1 23.3 ± 0.223.5 ± 0.4 23.8 ± 0.5

TABLE 9 Body weight of mice in the Tween 80:ethanol solution solutiongroups Control TPA 5 mg/kg 2.5 mg/kg 1.9 mg/kg 24.0 ± 0.4 23.6 ± 0.923.4 ± 0.4 23.3 ± 0.4 23.6 ± 0.1

TABLE 10 Effect of fluasterone on TPA-induced DNA synthesis Group cpm μgDNA/0.2 corrected cpm cpm/μg DNA Cremophor EL:ethanol Groups Control2,596 10.0 2,571 257.1 670 11.6 645 55.6  156.4 ± 142.5 TPA 54,343 35.754,308 1,521.2 54,744 36.9 54,709 1,482.6 1,501.9 ± 27.3   1.9 mg/kg5,990 28.7 5,965 207.8 4,050 25.1 4,025 160.4 187.1 ± 33.5 2.5 mg/kg7,093 16.7 7,068 423.2 6,268 15.2 6,243 410.7 6,366 13.7 6,341 462.8432.2 ± 27.2   5 mg/kg 1,902 15.0 1,877 125.1 3,145 14.0 3,120 222.92,770 13.3 2,745 206.4 184.8 ± 52.4 Tween/Ethanol Groups Control 327 5.4302 55.9 673 6.2 648 104.5  80.2 ± 34.4 TPA 4,301 17.5 4,276 244.3 3,13918.6 3,114 167.4 205.9 ± 54.4 1.9 mg/kg 3,139 14.4 3,114 216.3 3,17516.6 3,150 189.8 203.1 ± 18.7 2.5 mg/kg 670 7.0 645 92.1 1,912 7.5 1,887251.6 1,700 7.9 1,675 212.0 185.2 ± 83.0   5 mg/kg 1,716 7.8 1,691 216.81,631 4.8 1,606 334.6 2,561 4.6 2,536 551.3  367.6 ± 169.7

In the Cremophor EL:ethanol group, treatment with this formulationgreatly enhanced the effect of TPA in stimulating [³H] thymidineincorporation and epidermal DNA content when compared to the Tween80:ethanol group. Cremophor EL had been used to formulate the drugpaclitaxel for i.v. administration and is believed to cause acutehypersensitivity reactions, which may account for the enhanced TPAeffect with Cremophor EL in this experiment. Cremophor EL has beenreplaced by Tween-80 to formulate paclitaxel.

Example 6 The Effect of Topically Applied Fluasterone Dissolved inEthanol:Limonene:Isopropyl Myristate (81:15:4 v/v/v) or a Suspension ofMicronized Fluasterone in Ethanol:Water:Tween 80 (76:19:5 v/v/v) onTPA-Induced DNA Synthesis in Mouse Epidermis

Topical compositions of fluasterone with gel-like consistency wereprepared by the addition of carbomer 940 and triethanolamine to asuspension of micronized fluasterone in a mixture of ethanol:water:Tween80.

Both formulations suppress TPA-stimulated-epidermal DNA content per 2×2cm² of skin at a dose of 2.5 mg/kg. In the fluasterone-solution group,one of two mice in the 2.5 mg/kg dose group and both in the 5 mg/kggroup showed the typical artifactual increase in [³H] thymidine cpm/μgDNA. In the fluasterone-suspension group one mouse in the 5 mg/kg groupshowed this increase.

The data indicate that transdermal application of thefluasterone-suspension formulation is about as active as s.c.administration in suppressing TPA-stimulated epidermal hyperplasia. Thefluasterone-ethanol-limonene-isopropyl myristate solution is slightlymore active.

Methods and Results

Fluasterone Solution

The following were added to two separate glass scintillation vials:

-   -   8.2 mL absolute ethanol (PharmCo, ACS/USP Grade)    -   1.5 mL R+ limonene (Aldrich, 94%)    -   0.4 mL isopropyl myristate (Sigma, 98%)

To one scintillation vial 101.2 mg of micronized fluasterone was added.Upon vigorous shaking for a few minutes and fluasterone was dissolved.The other vial was used to treat Control and TPA-without-fluasteronemice.

Fluasterone Suspension

The following were added to two separate glass scintillation vials:

-   -   8 mL absolute ethanol (PharmCo, ACS/USP Grade)    -   2 mL double distilled water    -   0.5 mL Tween 80 (Sigma, ultra pure)

105.4 mg of micronized fluasterone was added to one vial. The suspensionwas sonicated with a Tekmar Tissumizer at a high setting of forapproximately three minutes. The non-fluasterone mixture was used forControl and TPA-without-fluasterone mice.

Treatment of Mice

Female CD-1 mice, 40-43 days old, were obtained from Charles RiverLaboratories, Kingston, N.Y. The mice were housed five per cage inplastic shoebox cages on Betachip bedding with 12 hours of alternatinglight and dark in the Central Animal Facility. The mice had ad libitumaccess to Purina 5015 chow and acidified tap water (pH 2.6). The miceremained in the Animal Facility for approximately one month.

The mice were shaved on the back and the abdomen one day prior totreatment. Only those mice showing no hair regrowth were used in theexperiment. The age of the mice at the time of the experiment wasapproximately 79 days of age. The usual age of mice used in priorexperiments was approximately 50 days of age.

The mice were treated as follows, with two mice in each experimentalgroup.

Control: The mice were anesthetized with Isoflurane, and then were givenan intramuscular injection of 0.1 mL solution of ketamine (50 mg/kg) andxylazine (10 mg/kg). Approximately 10 minutes after the i.m. injection(after the mice could not turn over when placed on their backs), themice were treated with 12.3 μL of ethanol:limonene:isopropyl myristatevehicle on the abdomen. The mice were anesthetized and on their backsfor approximately 30 minutes after application of vehicle. One hourafter the application of ethanol:limonene:isopropyl myristate solution,0.2 mL of acetone was applied to the shaved area on the back of eachmouse.TPA: The mice were anesthetized with Isoflurane, and then were given anintramuscular injection of 0.1 mL solution of ketamine (50 mg/kg) andxylazine (10 mg/kg). Approximately 10 minutes after the i.m. injection(after the mice could not turn over when placed on their backs), themice were treated with 12.3 μL of ethanol:limonene:isopropyl myristatesolution. The mice were anesthetized and on their backs forapproximately 30 minutes after application of vehicle. One hour afterthe application of ethanol:limonene:isopropyl myristate solution, 2 μgof TPA dissolved in 0.2 mL of acetone was applied to the shaved area onthe back of each mouse.1.5 mg/kg Fluasterone: The mice were anesthetized with Isoflurane, andthen were given an intramuscular injection of 0.1 mL solution ofketamine (50 mg/kg) and xylazine (10 mg/kg). Approximately 10 minutesafter the i.m. injection (after the mice could not turn over when placedon their backs), the mice were treated with 3.7 μL of a 10 mg/mLfluasterone:ethanol:limonene:isopropyl myristate solution to give a doseof 1.5 mg/kg. The mice were anesthetized and on their backs forapproximately 30 minutes after application offluasterone:ethanol:limonene:isopropyl myristate solution. One hourafter the application of fluasterone solution, 2 μg of TPA dissolved in0.2 mL of acetone was applied to the shaved area on the back of eachmouse.2.5 mg/kg Fluasterone The mice were anesthetized with Isoflurane, andthen were given an intramuscular injection of 0.1 mL solution ofketamine (50 mg/kg) and xylazine (10 mg/kg). Approximately 10 minutesafter the i.m. injection (after the mice could not turn over when placedon their backs), the mice were treated with 6.2 μL of a 10 mg/mLfluasterone:ethanol:limonene:isopropyl myristate solution to give a doseof 2.5 mg/kg. The mice were anesthetized and on their backs forapproximately 30 minutes after application offluasterone:ethanol:limonene:isopropyl myristate solution. One hourafter the application of fluasterone solution, 2 μg of TPA dissolved in0.2 mL of acetone was applied to the shaved area on the back of eachmouse.5 mg/kg Fluasterone The mice were anesthetized with Isoflurane, and thenwere given an intramuscular injection of 0.1 mL solution of ketamine (50mg/kg) and xylazine (10 mg/kg). Approximately 10 minutes after the i.m.injection (after the mice could not turn over when placed on theirbacks), the mice were treated with 12.3 μL of a 10 mg/mLfluasterone/ethanol:limonene:isopropyl myristate solution to give a doseof 5 mg/kg. The mice were anesthetized and on their backs forapproximately 30 minutes after application offluasterone:ethanol:limonene:isopropyl myristate solution. One hourafter the application of fluasterone solution, 2 μg of TPA dissolved in0.2 mL of acetone was applied to the shaved area on the back of eachmouse.Control: The mice were anesthetized with Isoflurane, and then were givenan intramuscular injection of 0.1 mL solution of ketamine (50 mg/kg) andxylazine (10 mg/kg). Approximately 10 minutes after the i.m. injection(after the mice could not turn over when placed on their backs), themice were treated with 12.3 μL of ethanol:water:Tween 80 solution on theabdomen. The mice were anesthetized and on their backs for approximately30 minutes after application of vehicle. One hour after the applicationof ethanol:water:Tween 80, 0.2 mL of acetone was applied to the shavedarea on the back of each mouse.TPA: The mice were anesthetized with Isoflurane, and then were given anintramuscular injection of 0.1 mL solution of ketamine (50 mg/kg) andxylazine (10 mg/kg). Approximately 10 minutes after the i.m. injection(after the mice could not turn over when placed on their backs), themice were treated with 12.3 μL of ethanol:water:Tween 80 solution. Themice were anesthetized and on their backs for approximately 30 minutesafter application of vehicle. One hour after the application ofethanol:water:Tween 80, 2 μg of TPA dissolved in 0.2 mL of acetone wasapplied to the shaved area on the back of each mouse.1.5 mg/kg Fluasterone The mice were anesthetized with Isoflurane, andthen were given an intramuscular injection of 0.1 mL solution ofketamine (50 mg/kg) and xylazine (10 mg/kg). Approximately 10 minutesafter the i.m. injection (after the mice could not turn over when placedon their backs), the mice were treated with 3.7 μL of a 10 mg/mLfluasterone:ethanol:water:Tween 80 suspension to give a dose of 1.5mg/kg. The mice were anesthetized and on their backs for approximately30 minutes after application of fluasterone suspension. One hour afterthe application of the fluasterone suspension, 2 μg of TPA dissolved in0.2 mL of acetone was applied to the shaved area on the back of eachmouse.2.5 mg/kg Fluasterone The mice were anesthetized with Isoflurane, andthen were given an intramuscular injection of 0.1 mL solution ofketamine (50 mg/kg) and xylazine (10 mg/kg). Approximately 10 minutesafter the i.m. injection (after the mice could not turn over when placedon their backs), the mice were treated with 6.2 μL of a 10 mg/mLfluasterone:ethanol:water:Tween 80 suspension to give a dose of 2.5mg/kg. The mice were anesthetized and on their backs for approximately30 minutes after application of fluasterone suspension. One hour afterthe application of the fluasterone suspension, 2 μg of TPA dissolved in0.2 mL of acetone was applied to the shaved area on the back of eachmouse.5 mg/kg Fluasterone The mice were anesthetized with Isoflurane, and thenwere given an intramuscular injection of 0.1 mL solution of ketamine (50mg/kg) and xylazine (10 mg/kg). Approximately 10 minutes after the i.m.injection (after the mice could not turn over when placed on theirbacks), the mice were treated with 12.3 μL of a 10 mg/mLfluasterone:ethanol:water:Tween 80 suspension to give a dose of 5 mg/kg.The mice were anesthetized and on their backs for approximately 30minutes after application of fluasterone suspension. One hour after theapplication of the fluasterone suspension, 2 μg of TPA dissolved in 0.2mL of acetone was applied to the shaved area on the back of each mouse.

The mice were sacrificed 20 hours after treatment by an overdose of CO₂.Twenty minutes prior to sacrifice, the mice were injected with 60 μCi of[³H] thymidine. The mice were treated with a depilatory to remove anyresidual hair. A piece of skin (approximately 2×2 cm²) was excised,placed in ice water for 30 seconds, then in 55° water for 30 seconds,then in ice water again for 30 seconds. The epidermis was scraped offusing a scalpel and the scrapings were placed into ice cold 0.4N TCA.The scrapings were homogenized using a Tekmar Tissumizer (80% power for30 seconds). The homogenates were centrifuged for 20 minutes at 3000×g.The precipitates were washed 4× with 0.2N TCA, and 2× with absoluteethanol. The DNA was hydrolyzed with 0.5N TCA for 30 minutes at 90°. Thetubes were centrifuged for 20 minutes at 3000×g. 0.2 mL aliquots of thesupernatants were counted in an LKB 1209 Rackbeta scintillation counter,using Scintiverse BD as counting medium. DNA content was determined bythe Burton diphenylamine assay.

TABLE 11 Effect of fluasterone on TPA-induced DNA synthesis Group cpm μgDNA/0.2 mL corrected cpm cpm/μg DNA Ethanol:Limonene:Isopropyl MyristateGroups Control 3,625 20.6 3,596 174.6 2,583 18.7 2,554 136.6 19.7 ± 1.34155.6 ± 26.9 TPA 11,138 34.5 11,109 322.0 8,574 30.8 8,545 277.4 32.7 ±2.61 299.7 ± 31.5 1.5 mg/kg 5,883 25.8 5,854 226.9 3,732 25.1 3,703147.5 25.5 ± 0.50 187.2 ± 56.1 2.5 mg/kg 3,453 18.1 3,424 189.2 7,10915.3 7,080 462.7 16.7 ± 1.98¹ 326.0 ± 193   5 mg/kg 8,496 15.9 8,467532.5 10,465 17.3 10,436 603.2 16.6 ± 0.99¹ 567.9 ± 50Ethanol:Water:Tween 80 Groups Control 2,327 18.3 2,298 125.6 1,662 23.71,633  68.9 21.0 ± 3.82 97.3 ± 40.1 TPA 7,303 34.9 7,274 208.4 6,58635.2 6,557 186.3 35.1 ± 0.21 197.4 ± 15.6 1.5 mg/kg 4,684 34.3 4,655135.7 3,510 35.2 3,481  98.9 34.8 ± 0.64 117.3 ± 26.0 2.5 mg/kg 2,17619.6 2,147 109.5 2,337 16.6 2,308 139.0 18.1 ± 2.1² 124.3 ± 20.9   5mg/kg 2,713 16.5 2,684 162.7 7,718 18.9 7,689 406.8 17.7 ± 1.7³ 284.8 ±173 Significantly less than corresponding TPA group. ¹p < 0.05, ²p <0.02, ³p < 0.01

Example 7 Effect of Dehydroepiandrosterone and Fluasterone onDexamethasone-Induced Thymic and Splenic Atrophy

Administration of dehydroepiandrosterone (DHEA, 60 mg/kg, s.c.) forthree days partially reverse thymic and splenic atrophy induced by asingle injection of dexamethasone (1.6 mg/mouse). In this experiment,the efficacy of either 10 mg/kg or 20 mg/kg DHEA administered s.c.versus the effect of 10 mg/kg or 20 mg/kg fluasterone was investigated.

Example 1 Efficacy of Subcutaneous Injection of Fluasterone AgainstThymic and Splenic Atrophy

Cremophor-saline vehicle: 5% Cremophor (polyethoxylated castor oil;Sigma Chemicals)-95% saline vehicle was prepared using 95 mL of sterile0.9% NaCl (filtered through sterile 0.22 micron filter) and 5 mL ofCremophor. The Cremophor was dissolved by swirling for approximately 5minutes.Dexamethasone: Dexamethasone solutions were prepared by dissolvingdexamethasone (Sigma Chemicals) in absolute ethanol. For example, 71.9mg of dexamethasone was dissolved in 2.9 mL of absolute ethanol. Inorder to prevent precipitation, the solution was kept on a hotplatebetween injections.

For the 10 mg/kg suspension, 25.7 mg of DHEA was weighed out,transferred to a glass scintillation vial, and 10.3 mL of theCremophor-saline vehicle was added. The DHEA suspension was homogenizedusing a Tissumizer at 80% power for 30 sec. The Tissumizer was rinsedwith 70% ethanol prior to homogenization. After the 10 mg/kg DHEAsuspension was homogenized the probe was rinsed 2× with 50 mL ofabsolute ethanol, then with 50 mL of 70% ethanol, and then dried withsterile gauze. For the 20 mg/kg DHEA suspension, 50.9 mg DHEA wassuspended in 10.2 mL of Cremophor-saline vehicle.

The probe was then washed with detergent, rinsed with 500 mL deionizedwater, 5×, rinsed with 70% ethanol, and then the probe was dried with asterile gauze pad prior to homogenizing the 10 mg/kg fluasteronesuspension.

19.4 mg of fluasterone was weighed out and transferred to a glassscintillation vial. 7.8 mL of Cremophor-saline vehicle was added to thevial and the contents of the vial were homogenized with a Tissumizer asdescribed previously. Prior to preparing the 20 mg/kg fluasterone, theprobe was rinsed 2× with 50 mL of absolute ethanol and 1× with 50 mL of70% ethanol. For the 20 mg/kg suspension, 47.0 mg was weighed out andtransferred to a glass scintillation vial. 9.4 mL of Cremophor-salinevehicle was added to the vial and the contents of the vial werehomogenized with a Tissumizer as described above.

Mice: Female CD-1 mice, 43-45 days old, were obtained from Charles RiverLaboratories. The mice were housed 5 per cage in plastic shoebox cageswith ad libitum access to Purina chow 5015 and water in the Pharmacy CAFwith 12 hr of alternating light and darkness.Pretreatment with DHEA or fluasterone: Prior to the experiment the micewere weighed, separated into 6 groups containing 5 mice each and treatedas follows:Control: Mice were treated s.c. with 0.1 mL of 5% Cremophor-95% salinefor three days. On the third day, the mice were also treated s.c. with0.06 mL of absolute ethanol.Dexamethasone: Mice were treated s.c. with 0.1 mL of 5% Cremophor-95%saline for three days. On the third day, the mice were also treated s.c.with 1.6 mg of dexamethasone dissolved in 0.06 mL of absolute ethanol.10 mg/kg DHEA: Mice were treated s.c. with 0.25 mg of DHEA suspended in0.1 mL of 5% Cremophor-95% saline for three days. On the third day, themice were also treated s.c. with 1.6 mg of dexamethasone dissolved in0.06 mL of absolute ethanol.20 mg/kg DHEA: Mice were treated s.c. with 0.5 mg of DHEA suspended in0.1 mL of 5% Cremophor-95% saline for three days. On the third day, themice were also treated s.c. with 1.6 mg of dexamethasone dissolved in0.06 mL of absolute ethanol.10 mg/kg Fluasterone: Mice were treated s.c. with 0.25 mg of fluasteronesuspended in 0.1 mL of 5% Cremophor-95% saline for three days. On thethird day, the mice were also treated s.c. with 1.6 mg of dexamethasonedissolved in 0.06 mL of absolute ethanol.20 mg/kg Fluasterone: Mice were treated s.c. with 0.5 mg of fluasteronesuspended in 0.1 mL of 5% Cremophor-95% saline for three days. On thethird day, the mice were also treated s.c. with 1.6 mg of dexamethasonedissolved in 0.06 mL of absolute ethanol.

All s.c. injections were made in the nape. All mice were lightlyanesthetized while the s.c. injections were done.

Twenty-four hours after dexamethasone treatment, the mice wereeuthanized by an overdose of isoflurane. The spleen and thymus wereremoved, cleaned of any adventitia, rinsed, blotted on filter paper, andweighed.

TABLE 12 Average thymus and spleen weights after euthanization BodyThymus Spleen Group Weight Thymus wt. wt/BW Spleen wt wt/BW CONTROL 24.6± 0.9 69.6 ± 8.8 2.8 ± 0.4 91.7 ± 7.4 3.7 ± 0.2 DEXAMETHASONE 25.2 ± 1.633.2 ± 1.3 1.3 ± 0.1 46.3 ± 2.5 1.9 ± 0.1 10 MG/KG DHEA 23.7 ± 1.0 31.7± 2.6 1.3 ± 0.2 46.4 ± 3.7 2.0 ± 0.2 20 MG/KG DHEA 24.5 ± 2.1 36.6 ± 5.21.5 ± 0.2 51.4 ± 2.2 2.1 ± 0.2 10 MG/KG 24.3 ± 1.0 60.9 ± 5.2 2.5 ± 0.3 77.5 ± 14.4 3.2 ± 0.5 FLUASTERONE 20 MG/KG 25.2 ± 0.6 60.8 ± 5.3 2.4 ±0.2  73.9 ± 13.8 2.9 ± 0.6 FLUASTERONE

The data indicate that DHEA, at a dose of 20 mg/kg, produces no apparentprotection against dexamethasone-induced thymic or splenic atrophy. Butsurprisingly, fluasterone is highly active in protecting thymus andspleen at a dose of 10 mg/kg.

Example 8 Comparison Between Micronized DHEA and Micronized Fluasterone

The dexamethasone induced thymic and splenic atrophy experiments wereperformed using procedures similar to those set forth above.

TABLE 13 Average body weight of mice before the experiment Dexa- 20mg/kg 60 mg/kg 10 mg/kg 20 mg/kg Control methasone DHEA DHEA FluasteroneFluasterone 26.1 ± 26.4 ± 1.6 26.1 ± 3.4 26.4 ± 3.3 26.7 ± 3.6 26.4 ±3.5 1.2

TABLE 14 Average mouse thymus and spleen weights after euthanizationBody Spleen Group Weight Thymus wt Thymus wt/BW Spleen wt wt/BW CONTROL26.6 ± 2.8 71.5 ± 7.0 2.7 ± 0.3 91.1 ± 11.3 3.4 ± 0.1 DEXAMETHASONE 27.3± 1   35.7 ± 3.8 1.3 ± 0.1 47.8 ± 3.3  1.7 ± 0.1 20 MG/KG DHEA 27.4 ±4.0 39.3 ± 4.1 1.4 ± 0.1 56.8 ± 8.1  2.1 ± 0.1 60 MG/KG DHEA 27.0 ± 3.163.2 ± 7.8 2.3 ± 0.2 79.9 ± 16.0 3.0 ± 0.4 10 MG/KG 26.7 ± 3.6  68.0 ±11.5* 2.5 ± 0.3  83.0 ± 6.2** 3.1 ± 0.3 FLUASTERONE 20 MG/KG 27.2 ± 3.1 66.3 ± 9.0** 2.4 ± 0.2 83.1 ± 7.9* 3.1 ± 0.4 FLUASTERONE *p < 0.01,versus 20 mg/kg DHEA group. **p < 0.001, versus 20 mg/kg DHEA group.

The data indicate that fluasterone is about 6× as potent as DHEA inprotecting against dexamethasone-induced thymic and splenic atrophy.

Example 9 Comparison of 30-methyl-16α-fluoro-5-androsten-17-one,16α-methyl-5-androsten-17-one, 30β-methyl-5-androsten-17-one,3β-methyl-16α-methyl-5-androsten-17-one and3β-methyl-16α-chloro-5-androsten-17-one with fluasterone and DHEA forAnti-Atrophic Activity

The dexamethasone induced thymic and splenic atrophy experiments wereperformed using procedures similar to those set forth above for a seriesof compounds of formula I.

Formulations containing one of the compounds of formula I,16α-fluoro-5-androsten-17-one (Fluasterone),3β-methyl-16α-fluoro-5-androsten-17-one, 16α-methyl-5-androsten-17-one,3β-methyl-5-androsten-17-one and,3β-methyl-16α-chloro-5-androsten-17-one,7α-hydroxy-16α-fluoro-5-androsten-17-one (7α-hydroxy-fluasterone) and3β-methyl-16α-methyl-5-androsten-17-one were prepared as describedbelow.

Fluasterone (10 mg/kg) suspension: For the 10 mg/kg Fluasteronesuspension, 35.4 mg non-micronized Fluasterone was weighed out,transferred to a glass scintillation vial, and 14.1 mL ofCremophor-saline vehicle was added. The suspension was homogenized usinga Tissumizer at 80% power for 30 sec. The Tissumizer was rinsed with 70%ethanol prior to homogenization.

The probe was then washed with detergent, rinsed with 500 mL deionizedwater, 5×, rinsed with 70% ethanol, and then the probe was dried with asterile gauze pad prior to homogenizing the 10 mg/kg3β-methyl-16α-fluoro-5-androsten-17-one suspension.

3β-methyl-16α-fluoro-5-androsten-17-one suspensions: For the 10 mg/kgsuspension, 11 mg of 3β-methyl-16α-fluoro-5-androsten-17-one was weighedout and transferred to a 7 mL plastic vial. 11 mL of Cremophor-salinevehicle was added to the vial and the contents of the vial werehomogenized with a Tissumizer as described previously. Prior topreparing the 20 mg/kg 3β-methyl-16α-fluoro-5-androsten-17-onesuspension, the probe was rinsed 2× with 50 mL of absolute ethanol and1× with 50 mL of 70% ethanol. For the 20 mg/kg suspension, 16.9 mg wasweighed out and transferred to a 7 mL plastic vial. 3.4 mL ofCremophor-saline vehicle was added to the vial and the contents of thevial were homogenized with a Tissumizer as described above.16α-methyl-5-androsten-17-one (10 mg/kg) suspension: The 10 mg/kgsuspension was prepared by weighing out 11.2 mg transferring to a 7 mLplastic vial, and adding 4.5 mL of vehicle. The suspension washomogenized as described previously.3β-methyl-16α-chloro-5-androsten-17-one suspensions: For the 10 mg/kgsuspension, 8.3 mg of 3β-methyl-16α-chloro-5-androsten-17-one wasweighed out, transferred to a 7 mL plastic vial, and 3.3 mL ofCremophor-saline vehicle was added. For the 20 mg/kg suspension, 17.4 mgwas weighed out, transferred to a 7 mL plastic vial, and 3.5 mL ofvehicle was added to the vial. The suspensions were homogenized asdescribed previously.3β-methyl-5-androsten-17-one suspensions The 10 mg/kg suspension wasprepared by weighing out 18.5 mg of 3β-methyl-5-androsten-17-one,transferring to a 7 mL plastic vial, and adding 7.4 mL of vehicle. Forthe 20 mg/kg suspension, 31 mg was weighed out and 6.2 mL of vehicle wasadded to the vial.3β-methyl-16α-methyl-5-androsten-17-one suspensions The 10 mg/kgsuspension was prepared by weighing out 20.0 mg suspension, transferringto a vial, and adding 8 mL of vehicle. For the 20 mg/kg suspension, 35.2mg was weighed out and added 7.0 mL of vehicle to the vial.Mice: Female CD-1 mice, 44-46 days old, were obtained from Charles RiverLaboratories. Prior to the experiments the mice were housed 5 per cagein plastic shoebox cages with ad libitum access to Purina chow 5015 andwater in the Pharmacy CAF with 12 hr of alternating light and darkness.

Two mice in the 20 mg/kg 3β-methyl-16α-methyl-5-androsten-17-one groupnever recovered from anesthesia. The other three mice in this group cameout of anesthesia very slowly.

Mice were treated with specific steroids and dexamethasone-inducedthymic and splenic atrophy procedures were performed as set forth above.

TABLE 15 Average thymus and spleen weights of mice after euthanizationSpleen Group Body Weight Thymus wt Thymus wt/BW Spleen wt wt/BW CONTROL25.9 ± 1.1 83.2 ± 6.6 3.2 ± 0.3 98.5 ± 9.7 3.8 ± 0.3 DEXAMETHASONE 25.2± 0.9 36.0 ± 4.9 1.4 ± 0.2 47.2 ± 6.1 1.9 ± 0.2 10 MG/KG 24.8 ± 0.8 67.5± 3.9 2.7 ± 0.2 80.0 ± 7.7 3.2 ± 0.4 FLUASTERONE 10 mg/kg 25.5 ± 0.743.8 ± 3.0 1.7 ± 0.1 50.9 ± 7.5 2.0 ± 0.3 3β-methyl-16α-fluoro-5-androsten-17-one 20 mg/kg 25.5 ± 0.9 55.8 ± 5.4 2.2 ± 0.2 55.0 ± 4.6 2.2± 0.1 3β-methyl-16α-fluoro-5- androsten-17-one 10 mg/kg 25.3 ± 0.8 28.0± 1.7 1.1 ± 0.1 37.8 ± 6.1 1.5 ± 0.3 16α-methyl-5- androsten-17-one 10mg/kg 24.2 + 1.1 61.9 + 3.9 2.6 + 0.2 70.7 + 4.5 2.9 + 0.23β-methyl-5-androsten- 17-one 20 mg/kg 25.8 ± 2.6 62.2 ± 4.1 2.4 ± 0.2 67.0 ± 11.3 2.6 ± 0.2 3β-methyl-5-androsten- 17-one 10 mg/kg 25.8 ± 0.642.6 + 4.7 1.7 + 0.2 44.9 + 9.2 1.7 + 0.3 3β-methyl-16α-methyl-5-androsten-17-one 20 mg/kg 25.3 ± 1.6 37.7 ± 4.0 1.5 ± 0.2 44.4 ± 8.71.8 ± 0.3 3β-methyl-16α-methyl- 5-androsten-17-one 10 mg/kg 26.0 ± 1.931.7 ± 4.1 1.2 ± 0.1 42.3 ± 5.6 1.6 ± 0.2 3β-methyl-16α-chloro-5-androsten-17-one 20 mg/kg 26.4 ± 0.7 28.6 ± 3.2 1.1 ± 0.1 38.6 ± 6.31.2 ± 0.3 3β-methyl-16α-chloro-5- androsten-17-one

The data indicate that fluasterone is the most potent compound inprotecting against dexamethasone-induced thymic and splenic atrophy.

Example 10 Comparison of 16α-fluoro-5α-androstan-17-one (8356),7α-hydroxyfluasterone, 16α-fluoro-5-androsten-17-ol,16α-hydroxy-5-androsten-17-one with Fluasterone and DHEA forAnti-Atrophic Activity

The activity of title compounds against dexamethasone-induced thymic andspleen atrophy was tested in procedures analogous to those describedabove, and the results are given in Table 15.

TABLE 16 Average thymus and spleen weights of mice after euthanizationBody Thymus Spleen Group Weight Thymus wt wt/BW Spleen wt wt/BW CONTROL25.6 ± 1.0  78.8 ± 11.2 3.1 ± 0.4 85.8 ± 20.1 3.3 ± 0.7 DEXAMETHASONE24.7 ± 1.2 27.3 ± 3.3 1.1 ± 0.2 42.8 ± 4.6  1.7 ± 0.2 20 MG/KG 8356 25.2± 2.9 40.8 ± 5.8 1.6 ± 0.1 54.0 ± 4.5  2.0 ± 0.3 40 MG/KG 8356 25.2 ±1.4 54.4 ± 4.5 2.2 ± 0.2 51.2 ± 3.0  2.0 ± 0.2 5 MG/KG 25.7 ± 1.7  50.8± 10.9 2.0 ± 0.3 62.0 ± 10.2 2.4 ± 0.3 FLUASTERONE 10 MG/KG FLUASTERONE25.2 ± 1.4 65.6 ± 4.4 2.6 ± 0.2 89.4 ± 12.3 3.5 ± 0.4 5 mg/kg 24.5 ± 1.043.8 ± 6.9 1.8 ± 0.3 46.3 ± 11.1 1.8 ± 0.5 7α-hydroxyfluasterone 10mg/kg 24.0 ± 1.3  54.8 ± 12.1 2.3 ± 0.4 50.9 ± 12.5 2.1 ± 0.57α-hydroxyfluasterone 20 mg/kg 25.1 ± 1.0 42.3 ± 4.6 1.7 ± 0.2 52.3 ±10.8 2.1 ± 0.4 16α-fluoro-5-androsten-17-ol 60 mg/kg 25.7 ± 1.5 45.1 ±5.1 1.8 ± 0.2 45.9 ± 5.1  1.8 ± 0.2 16α-fluoro-5-androsten-17-ol 20mg/kg 25.4 ± 1.5 41.2 ± 1.3 1.6 ± 0.1 46.6 ± 7.5  1.8 ± 0.216α-hydroxy-5-androsten-17- one 60 mg/kg 23.8 ± 0.8 45.9 ± 3.1 1.9 ± 0.253.6 ± 6.3  2.2 ± 0.3 16α-hydroxy-5-androsten-17- one

Example 11 Gel Compositions with Fluasterone

A gel can be formed upon the addition of small amounts of water (about5%) to 10 mg/mL solutions of fluasterone in Tween 80:ethanol. However,substantial fluasterone precipitation occurred. An attempt was made toform a gel by adding carbomer to mixtures of ethanol and various organicsolvents without water, and this was unsuccessful.

A gel containing fluasterone was made using a suspension of micronizedfluasterone. This suspension of fluasterone was about as effective ass.c.-injection in abolishing TPA-stimulated DNA synthesis in mouse.

Fluasterone:Ethanol:Water:Tween Gel

The following were added to a screw cap glass scintillation vial:

7.5 mL absolute ethanol (PharmCo, ACS/USP Grade)

2.5 mL double distilled water

0.5 mL Tween 80 (Sigma, ultra pure)

97.1 mg of micronized fluasterone was added to the above and thesuspension was sonicated with a Tekmar Tissumizer for about one minuteat high speed. 60 mg Carbomer 940 (Spectrum Chem.) was added to thesuspension and shook vigorously for several minutes, followed by theaddition of 10 μL of triethanolamine (Sigma. Suspension turned into agel after overnight incubation at room temperature. The gel was easy toapply and disappeared quickly from the skin.

Example 12 Micronized Fluasterone

A 450 gm sample of unmilled fluasterone (19.33 μm) was processed throughan air jet pulverizer mill at 90 psi at rate of6 kg/hr. The resultingmilled fluasterone had a mean particle size of 6.16 μm (Micronized I;MI).

A 350 gm of unmilled fluasterone was processed through an air-jetpulverizer at 90 psi at a reduced rate of 0.60 kg/hr, resulting in finerparticles. This procedure produced a milled fluasterone product with amean particle size of 2.04 μm. (Micronized II; MII)

Reducing fluasterone mean particle size from 19.33 to 6.16 μm enhancedoral bioavailability, and reducing particle size to 2.04 μm furtherimproved oral bioavailability.

Determination of Oral Bioavailability of Micronized Fluasterone:

Treatment of Mice: Mice CD-1 mice (42-45 days old) were obtained fromCharles River Laboratories, Kingston, N.Y. The mice were housed threeper cage in plastic shoebox cages on corncob bedding at 72°±2° F. with50%±5% humidity and 12 hours of alternating light and dark in the FelsAnimal Facility. The mice had ad libitum access to Purina 5015 chow andacidified tap water (pH≦2.6). The mice were allowed to acclimate to thefacility for one week prior to use in an experiment.102.7 mg of Micronized fluasterone I was weighed using a Mettler AE-50balance, transferred to a glass scintillation vial, and homogenized in6.0 mL of carboxymethylcellulose-Tween 80 using a Tekmar Tissumizer (200mg/kg micronized I suspension, 200 MI). 88.4 mg of micronizedfluasterone II was then weighed, transferred to a glass scintillationvial, and homogenized in 5.2 mL of CMC-Tween using a Tekmar Tissumizer(200 mg/kg micronized II suspension, 200 MII). 179.2 mg of micronizedfluasterone II was weighed, transferred to a glass scintillation vial,and homogenized in 5.2 mL of CMC-Tween (400 mg/kg micronized IIsuspension, 400 MII). 93.0 mg of non-micronized fluasterone (meanparticle size, 19.92 μm) was weighed, transferred to a glassscintillation vial, and homogenized in 5.5 mL of CMC-Tween (200 mg/kgnon-micronized suspension, 200 R). 167.0 mg of non-micronizedfluasterone was weighed, transferred to a glass scintillation vial, andhomogenized in 4.9 mL of CMC-Tween (400 mg/kg non-micronized suspension,400 R). 261.5 mg of non-micronized fluasterone was weighed, transferredto a glass scintillation vial, and homogenized in 5.1 mL of CMC-Tween(600 mg/kg non-micronized suspension, 600 R). A magnetic stirrer wasadded to each of the vials and the suspensions were stirred forapproximately 45 minutes prior to intubation. The mice were intubatedusing 18 gauge stainless steel intubating needles.The mice were treated as follows:Control were intubated with 0.25 ml of CMC-Tween. One hour afterintubation with CMC-Tween, the mice were treated with 0.2 mL of acetone.The mice were given ad libitum access to chow immediately afterintubation.TPA were intubated with 0.25 ml of CMC-Tween. One hour after intubationwith CMC-Tween, the mice were treated with 2 μg of TPA in 0.2 mL ofacetone. The mice were given ad libitum access to chow immediately afterintubation.600 R were intubated with 0.25 ml of a 600 mg/kg suspension ofnon-micronized fluasterone. One hour after intubation with CMC-Tween,the mice were treated with 2 μg of TPA in 0.2 mL of acetone. The micewere given ad libitum access to chow immediately after intubation.400 R were intubated with 0.25 ml of a 400 mg/kg suspension ofnon-micronized fluasterone. One hour after intubation with CMC-Tween,the mice were treated with 2 μg of TPA in 0.2 mL of acetone. The micewere given ad libitum access to chow immediately after intubation.200 R were intubated with 0.25 ml of a 20 mg/kg suspension ofnon-micronized fluasterone. One hour after intubation with CMC-Tween,the mice were treated with 2 μg of TPA in 0.2 mL of acetone. The micewere given ad libitum access to chow immediately after intubation.400 MII were intubated with 0.25 ml of a 400 mg/kg suspension ofmicronized fluasterone II in CMC-Tween. One hour after intubation withCMC-Tween, the mice were treated with 2 μg of TPA in 0.2 mL of acetone.The mice were given ad libitum access to chow immediately afterintubation.200 MII were intubated with 0.25 ml of a 200 mg/kg suspension ofmicronized fluasterone II in CMC-Tween. One hour after intubation withCMC-Tween, the mice were treated with 2 μg of TPA in 0.2 mL of acetone.The mice were given ad libitum access to chow immediately afterintubation.200 MI were intubated with 0.25 ml of a 200 mg/kg suspension ofmicronized fluasterone I in CMC-Tween. One hour after intubation withCMC-Tween, the mice were treated with 2 μg of TPA in 0.2 mL of acetone.The mice were given ad libitum access to chow immediately afterintubation.

All mice started to consume chow immediately after the chow was added tocage.

The mice were sacrificed 20 hours after treatment by an overdose of CO₂.Twenty minutes prior to sacrifice, the mice were injected with 60 μCi of[3H]thymi

dine (Amersham). The mice were treated with a depilatory to remove anyresidual hair. A 2×2 cm piece of skin was excised, placed in ice waterfor 30 seconds, then in 55° water for 30 seconds, then in ice wateragain for 30 seconds. The epidermis was scraped off using a scalpel andthe scrapings were placed into ice cold 0.4N TCA. The scrapings werehomogenized using a Tekmar Tissumizer (80% power for 30 seconds). Thehomogenates were centrifuged for 20 minutes at 3000×g. The precipitateswere washed 4× with 0.2N TCA, and 2× with absolute ethanol. The DNA washydrolyzed with 0.5N TCA for 30 minutes at 90°. The tubes werecentrifuged for 20 minutes at 3000×g. A 0.2 ml aliquot of eachhydrolysate was counted in a LKB Rackbeta scintillation counter usingScintiverse II as the counting medium. DNA content was determined by theBurton diphenylamine assay.

TABLE 17 Average Body Weights Control TPA 600 R 400 R 200 R 400 MII 200MII 200 MI 21.3 ± 0.4 21.4 ± 0.5 21.3 ± 0.3 21.2 ± 0.5 21.2 ± 0.4 21.5 ±0.2 21.3 ± 0.3 21.3 ± 0.4

TABLE 18 Effect of Fluasterone on TPA-Induced Epidermal DNA SynthesisGroup corrected cpm μg DNA/0.2 ml cpm/μg DNA Control 1,900 16.4 115.92594 14.6 177.7 1494 18.0  83.0 125.5 ± 48.1 TPA 8,699 28.2 308.5 9,87728.0 351.6 8,235 31.9 258.6 306.1 ± 46.8 600 R 2,343 17.9 130.9 600mg/kg 2,346 17.3 135.6 NON-MICRONIZED 2,732 16.5 165.6 144.0 ± 18.8 400R 3,479 21.4 162.9 400 mg/kg 4,101 18.2 225.3 NON-MICRONIZED 3,358 19.7170.4 186.1 ± 34.2 200 R 5,120 20.0 256.0 200 mg/kg 7,807 18.0 433.7NON-MICRONIZED 5,365 19.7 257.9  315.9 ± 102.1 400 MII 1,941 16.5 117.6400 mg/kg 2,136 17.2 120.7 MICRONIZED II 1,138 17.7 120.8 119.7 ± 1.8 200 MII 2,666 16.0 166.6 200 mg/kg 2,856 23.1 123.6 MICRONIZED II 2,75322.9 120.2 136.8 ± 25.9 200 MI 2,763 17.7 156.1 200 mg/kg 3,554 24.4145.7 MICRONIZED I 3,563 23.5 151.6 (first prep) 151.1 ± 5.2 The Micronized I sample (6.16 μm) was about 2 to 3-fold more potent thanthe Non-micronized sample (19.33 μm), and the Micronized II sample (2.14μm) was more potent than Micronized I.

Example 13 Effect of Orally Administered Micronized Fluasterone andNanosized Fluasterone on TPA-Induced Epidermal DNA Synthesis in Mice

In this experiment nanosized fluasterone (mean particle size=0.621 μm)was compared to micronized fluasterone (mean particle size 5.5 μm) forefficacy in suppressing TPA-stimulated epidermal DNA synthesis in mouseskin. Both preparations were administered as suspensions in an aqueousvehicle at p.o. dosages of 200 mg/kg, 150 mg/kg, and 100 mg/kg.

Mice were given an oral intubation of 0.2 ml sesame oil ten minutesprior to dosing with fluasterone in aqueous suspension. In priorexperiments we found that either dosing with 0.2 ml sesame oil orallowing the mice to consume food shortly before administeringfluasterone in an aqueous vehicle enhances bioavailability. Presumablythis had the same effect (i.e. bile salt release) as food given topatients as shown in the phase I study.

The nanosized fluasterone suspension is the most active formulation wehave tested to date and would appear to be about 2× as bioavailable asthe 5.5 μm suspension.

Methods and Experimental Results

Female CD-1 mice were obtained from Charles River Laboratories,Wilmington, Mass. at 46-48 days of age. The mice were housed three percage in plastic shoebox cages on Aspen chip bedding with 12 hours ofalternating light and dark in the Fels Animal Facility. The mice had adlibitum access to Purina 5015 chow and acidified tap water (pH #2.6).The mice were allowed to acclimate to the facility for one week prior touse in an experiment. Three days later the mice were shaved.

The concentration of the micronized fluasterone suspension was 155.4mg/g of suspension while that of the nanosized fluasterone suspensionwas 109.5 mg/g of suspension. A gram of the micronized suspension wasequal to 1.02 ml, while that of the nanosized suspension was assumed tobe 1 g/ml.

Five suspensions were prepared approximately one-half hour prior totreatment. 354 μL (53.9 mg) of micronized fluasterone was added to 1,646μL of vehicle to give a suspension yielding a dose of 200 mg/kg whengiven p.o. at 0.2 mL (26.9 mg/ml). 263 μL of micronized suspension (40.1mg) was added to 1,737 μL of vehicle to give a suspension yielding 150mg/kg (20.0 mg/ml). For the nanosized suspensions: 473 μL (51.8 mg) wasadded to 1,527 μL of vehicle to give a suspension yielding 200 mg/kg(25.9 mg/ml), 360 μL (39.4 mg) of nanosized fluasterone suspension wasadded to 1,640 μL of vehicle to give a suspension yielding 150 mg/kg(19.7 mg/ml), 300 μL (32.9 mg) was added to 1,700 μL of vehicle to givea suspension yielding 125 mg/kg (16.6 mg/ml) and 230 μL (25.2 mg) wasadded to 1,770 μL of vehicle to give a suspension yielding 100 mg/kg(12.6 mg/ml). A magnetic stirrer was added to each of the vials and thesuspensions were stirred without heat.

The mice were treated as follows:

-   Control The mice were given 0.2 ml of sesame oil p.o, and ten    minutes later, were treated with 0.2 ml of suspension vehicle p.o.    One hour after treatment with the vehicle, the mice were treated    topically with 0.2 ml of acetone.-   TPA The mice were given 0.2 ml of sesame oil p.o, and ten minutes    later, were treated with 0.2 ml of suspension vehicle p.o. One hour    after treatment with the vehicle, the mice were treated topically    with 2 μg of TPA dissolved in 0.2 ml of acetone.-   200 mg/kg Fluasterone Micronized    -   The mice were treated 0.2 ml of sesame oil p.o. ten minutes        prior to treatment p.o. with 0.2 ml of a suspension of        micronized fluasterone which gave a dose of 200 mg/kg. One hour        after treatment with the micronized fluasterone suspension, the        mice were treated topically with 2 μg of TPA dissolved in 0.2 ml        of acetone.-   150 mu/kg Fluasterone Micronized The mice were treated 0.2 ml of    sesame oil p.o. ten minutes prior to treatment p.o. with 0.2 ml of a    suspension of micronized fluasterone which gave a dose of 150 mg/kg.    One hour after treatment with the micronized fluasterone suspension,    the mice were treated topically with 2 μg of TPA dissolved in 0.2 ml    of acetone.-   200 mu/kg Fluasterone Nanosized The mice were treated 0.2 ml of    sesame oil p.o. ten minutes prior to treatment p.o. with 0.2 ml of a    suspension of nanosized fluasterone which gave a dose of 200 mg/kg.    One hour after treatment with the nanosized fluasterone suspension,    the mice were treated topically with 2 μg of TPA dissolved in 0.2 ml    of acetone.-   200 mu/kg Fluasterone Nanosized The mice were treated 0.2 ml of    sesame oil p.o. ten minutes prior to treatment p.o. with 0.2 ml of a    suspension of nanosized fluasterone which gave a dose of 150 mg/kg.    One hour after treatment with the nanosized fluasterone suspension,    the mice were treated topically with 2 μg of TPA dissolved in 0.2 ml    of acetone.-   125 mu/kg Fluasterone Nanosized The mice were treated 0.2 ml of    sesame oil p.o. ten minutes prior to treatment p.o. with 0.2 ml of a    suspension of nanosized fluasterone which gave a dose of 150 mg/kg.    One hour after treatment with the nanosized fluasterone suspension,    the mice were treated topically with 2 μg of TPA dissolved in 0.2 ml    of acetone.-   100 mu/kg Fluasterone Nanosized The mice were treated 0.2 ml of    sesame oil p.o. ten minutes prior to treatment p.o. with 0.2 ml of a    suspension of nanosized fluasterone which gave a dose of 100 mg/kg.    One hour after treatment with the nanosized fluasterone suspension,    the mice were treated topically with 2 μg of TPA dissolved in 0.2 ml    of acetone.

The mice were sacrificed 20 hours after treatment by an overdose of CO₂.Twenty minutes prior to sacrifice, the mice were injected with 60 μCi of[³H]thymidine (Amersham). The mice were treated with a depilatory toremove any residual hair. A 2×2 cm² piece of skin was excised, placed inice water for 30 seconds, then in 55EC water for 30 seconds, then in icewater again for 30 seconds. The epidermis was scraped off using ascalpel and the scrapings were placed into ice cold 0.4N TCA. Thescrapings were homogenized using a Tekmar Tissumizer (80% power for 30seconds). The homogenates were centrifuged for 20 minutes at 3,000×g.The precipitates were washed 4× with 0.2N TCA, and 2× with absoluteethanol. The DNA in each sample was hydrolyzed with 0.5N TCA for 30minutes at 90E. The tubes were centrifuged for 20 minutes at 3000×g. A0.2 ml aliquot of each hydrolysate was counted in a LKB Rackbetascintillation counter using Scintiverse II BD as the counting medium.DNA content was determined by the Burton diphenylamine assay.

TABLE 19 Body Weights 200 mg/kg 150 mg/kg 200 mg/kg 150 mg/kg 125 mg/kg100 mg/kg Control TPA micronized micronized nanosized nanosizednanosized nanosized 26.0 ± 1.9 26.8 ± 3.4 26.9 ± 1.5 26.7 ± 2.1 25.9 ±1.6 26.2 ± 1.6 26.5 ± 1.2 25.2 ± 1.0

TABLE 20 Results: Effect of oral intake of micronized and nanosizedfluasterone Group μg DNA cpm/μg DNA dpm/μg DNA Control 8.0 ± 0.9 161.4 ±9.6  260.4 ± 15.5 TPA 13.9 ± 0.3  374.1 ± 16.8 603.4 ± 36.8 Micronized200 mg/kg 7.0 ± 0.3 163.9 ± 26.1 264.4 ± 42.1 Micronized 150 mg/kg 14.4± 0.5  479.3 ± 41.5 773.1 ± 66.8 Nanosized 200 mg/kg 8.1 ± 0.8 102.2 ±26.7 164.7 ± 43.0 Nanosized 150 mg/kg 6.9 ± 0.3 123.5 ± 10.6 199.3 ±17.1 Nanosized 125 mg/kg 7.1 ± 0.4 125.0 ± 29.7 201.6 ± 47.9 Nanosized100 mg/kg 8.9 ± 0.6 187.4 ± 41.1 302.2 ± 66.2

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A pharmaceutical composition comprising asuspension comprising a nanosized compound selected from the groupconsisting of 3β-methyl-5-androsten-17-one,3β-methyl-16α-fluoro-5-androsten-17-one and16α-fluoro-5-androsten-17-one; a lower alkyl alcohol; a surfactant; andoptionally, a long chain alcohol.
 2. A composition according to claim 1,wherein said long chain alcohol corresponds to the formulaCH₃(CH₂)_(n)—OH, wherein n is an integer in the range of 9-24.
 3. Acomposition according to claim 2, wherein said long chain alcohol isselected from the group consisting of decyl alcohol, cetyl alcohol,stearyl alcohol, lauryl alcohol, myristyl alcohol, oleyl alcohol andmixtures thereof.
 4. A composition according to claim 1, furthercomprising water.
 5. A transdermal delivery system comprising acomposition of claim
 1. 6. A topical formulation comprising acomposition of claim
 1. 7. A method for the treatment of obesity, typeII diabetes, arthritis or metabolic syndrome in a patient in needthereof, which comprises administering to said patient a therapeuticallyeffective amount of a composition of claim
 1. 8. The method according toclaim 7, wherein said treatment is for type II diabetes.
 9. The methodaccording to claim 7, wherein the treatment is for obesity.
 10. A gelcomprising a nanosized compound selected from the group consisting of3β-methyl-5-androsten-17-one, 3β-methyl-16α-fluoro-5-androsten-17-oneand 16α-fluoro-5-androsten-17-one; a lower alkyl alcohol; water; asurfactant; a thickening agent; and optionally a base.
 11. The gelaccording to claim 10, wherein said lower alkyl alcohol is selected fromthe group consisting of ethanol, methanol, butanol, pentanol,isopropanol and n-propanol.
 12. The gel according to claim 10, whereinsaid base is selected from the group consisting of triethanolamine,diethanolamine and triethylamine.
 13. A method of preparing a gelcomprising a nanosized compound selected from the group consisting of3β-methyl-5-androsten-17-one, 3β-methyl-16α-fluoro-5-androsten-17-oneand 16α-fluoro-5-androsten-17-one, comprising the steps of: mixing alower alkyl alcohol, a surfactant, a base, water and said nanosizedcompound; adding and mixing a cross-linked acrylic acid polymer; andincubating said ingredients until gel formation.
 14. A method oftreating cancer comprising administering to a subject in need thereof acomposition of claim
 1. 15. The method of claim 14, wherein said canceris prostate cancer.
 16. The gel according to claim 10 wherein thesurfactant is a polysorbate.
 17. The gel according to claim 16 whichcomprises: from about 30 to about 90% (v/v) lower alcohol; and fromabout 0.01 to about 5% (v/v) surfactant.
 18. A method of treatingdiabetes comprising administering to a mammal in need thereof acomposition according to claim
 1. 19. The method according to claim 7,wherein said treatment is for arthritis.
 20. A transdermal deliverysystem comprising a composition of claim
 10. 21. A topical formulationcomprising a composition of claim
 10. 22. A method for the treatment ofobesity, arthritis, type II diabetes or metabolic syndrome in a patientin need thereof, which comprises administering to said patient atherapeutically effective amount of a composition comprising a gelaccording to claim
 10. 23. The method according to claim 22, whereinsaid treatment is for type II diabetes.
 24. The method according toclaim 22 wherein the treatment is for obesity.
 25. The method accordingto claim 22, wherein the treatment is for rheumatoid arthritis orosteoarthritis.
 26. The method according to claim 22, wherein said loweralkyl alcohol is selected from the group consisting of ethanol,methanol, butanol, pentanol, isopropanol and n-propanol.
 27. The methodaccording to claim 22 wherein the surfactant is a polysorbate.
 28. Themethod according to claim 27, wherein said polysorbate is selected fromthe group consisting of polyoxyethylene-20-sorbitan monooleate (Tween80), polyoxyethylene-20-sorbitan monostearate (Tween 60),polyoxyethylene-20-sorbitan monopalmitate (Tween 40),polyoxyethylene-20-sorbitan monolaurate (Tween 20), and mixturesthereof.
 29. The method according to claim 13, wherein said cross-linkedacrylic acid polymer is a Carbomer.
 30. The composition of claim 1,wherein said nanosized compound is 16α-fluoro-5-androsten-17-one. 31.The method of claim 7 or 22, wherein said nanosized compound is16α-fluoro-5-androsten-17-one.
 32. The gel according to claim 1 whereinsaid nanosized compound is 16α-fluoro-5-androsten-17-one.
 33. The methodof preparing a gel according to claim 13 wherein the nanosized compoundis 16α-fluoro-5-androsten-17-one.
 34. The pharmaceutical compositionaccording to claim 1 wherein said surfactant is a polysorbate or apolyethyleneglycol substituted fatty acid.
 35. The pharmaceuticalcomposition according to claim 34 wherein said polysorbate ispolyoxyethylene-20-sorbitan monooleate (Tween 80), and wherein saidpharmaceutical composition comprises a lower alkyl alcohol in the rangeof from about 30 to about 90% (v/v), polyoxyethylene-20-sorbitanmonooleate (Tween 80) in the range of from about 0.01% to about 3.5% andwater in the range of from about 0% to about 60%.
 36. The methodaccording to claim 14, wherein said cancer is a cancer of the mammarygland, skin, colon, liver or lymphatic system.
 37. A compositionaccording to claim 34, wherein said polysorbate is selected from thegroup consisting of polyoxyethylene-20-sorbitan monooleate (Tween 80),polyoxyethylene-20-sorbitan monostearate (Tween 60),polyoxyethylene-20-sorbitan monopalmitate (Tween 40),polyoxyethylene-20-sorbitan monolaurate (Tween 20), polyethyleneglycolstearate, polyethyleneglycol oleate and mixtures thereof.
 38. The methodaccording to claim 19, wherein the treatment is for rheumatoid arthritisor osteoarthritis.
 39. The gel according to claim 16, wherein saidpolysorbate is selected from the group consisting ofpolyoxyethylene-20-sorbitan monooleate (Tween 80),polyoxyethylene-20-sorbitan monostearate (Tween 60),polyoxyethylene-20-sorbitan monopalmitate (Tween 40),polyoxyethylene-20-sorbitan monolaurate (Tween 20), and mixturesthereof.